Method for preparing Mucuna pruriens seed extract

ABSTRACT

The present invention provides pharmaceutical compositions comprising  Mucuna pruriens  seeds or one or more  Mucuna pruriens  seed components, substances, fractions or mixtures or substances obtained therefrom. Furthermore, the invention relates to the use of  Mucuna pruriens  seed powder or one or more  Mucuna pruriens  components, substances, fractions or mixtures or substances obtained therefrom for the preparation of a pharmaceutical composition for preventing, alleviating or treating neurological diseases. Additionally, the invention relates to the use of  Mucuna pruriens  seeds for the preparation of a pharmaceutical composition for neuroprotection or neurostimulation and to methods of preparing extracts of  Mucuna pruriens  which can be used for the preparation of a pharmaceutical composition for treating neurological diseases. Finally, the invention relates to the use of  Mucuna pruriens  seeds for the preparation of a pharmaceutical composition for the treatment of Parkinson&#39;s Disease to obtain a broader therapeutic window in L-Dopa therapy, to delay a need for combination therapy, to obtain an earlier onset and longer duration of L-Dopa efficacy, and to prevent or alleviate acute and chronic L-Dopa toxicity.

The present invention provides pharmaceutical compositions comprisingMucuna pruriens seeds or one or more Mucuna pruriens seed components,substances, fractions or mixtures or substances obtained therefrom.Furthermore, the invention relates to the use of Mucuna pruriensseedpowder or one or more Mucuna pruriens components, substances,fractions or mixtures or substances obtained therefrom for thepreparation of a pharmaceutical composition for preventing, alleviatingor treating neurological diseases. Additionally, the invention relatesto the use of Mucuna pruriens seeds for the preparation of apharmaceutical composition for neuroprotection or neurostimulation andto methods of preparing extracts of Mucuna pruriens which can be usedfor the preparation, of a pharmaceutical composition for treatingneurological diseases.

Several documents are cited throughout the text of this specification.The disclosure content of the documents cited herein (including anymanufacture's specifications, instructions, etc.) is herewithincorporated by reference.

A large number of neurological and neurological degenerative disease areknown, many of which are presently not curable. These diseases comprisemedical conditions such as Parkinson's disease, Chorea Huntington,Hallervorder-Spatz disease, Alzheimer's disease, senile dementia,Creutzfeldt-Jakob disease, artheriosclerotic dementia, cerebralthrombangitis obliterans and many others. Parkinson's disease (PD) is aprogressive movement and age-related disorder that is estimated toaffect for example more than 500,000 persons in the United States, withas many as 50,000 new cases each year, at an estimated cost of 27billion dollars annually. Usually PD begins in a person's late 50s orearly 60s, it causes a progressive decline in movement control,affecting the ability to control initiation, speed and smoothness ofmotion. Symptoms of PD are seen in up to 15% of those between the ages65 to 74, and almost 30% of those between the ages of 75 and 84. PD isone of the best characterized diseases of the basal ganglia. Thesymptoms that come along with the disease are a rhythmical tremor atrest, a unique increase in muscle tone or rigidity that has oftencogwheel- or ratchet-like characteristic, difficulty in the initiationof movement and paucity of spontaneous movements (akinesia), andslowness in the execution of movement (bradykinesia). In humanssuffering from Parkinson's disease dopamine is missing or mostdrastically reduced in certain regions of the brain which areessentially needed, for example, for controlling the movement of thebody. L-DOPA is metabolised within the body to dopamine which plays anoutstanding role in the metabolism of the brain as neurotransmitter.

Beside the reduction of dopamine which coincides with the symptoms ofthe disease, it is also speculated that the destruction ofdopamine-producing nerve cells especially those of the substantia nigrapars compacta in the mid brain (one of the principal movement controlcenters in the brain) contributes to the disease. This control centerhelps to refine movement patterns throughout the body. It was observedthat the brains of patients with Parkinson's disease also have loss ofnerve cells and depigmentation in the two pigmented loci of the brainstem: the substantia nigra and the locus ceruleus. Thereby, the severityof changes in the substantia nigra parallels the reduction of dopaminein the striatum. Because the pars compacta of the substantia nigracontains many of the dopaminergic nerve cell bodies in the brain, theseobservations suggest that the dopaminergic pathway from the substantianigra to the striatum is disturbed in Parkinson's disease. However, themolecular mechanisms underlying PD are still under investigation andpoorly understood.

From the above-mentioned findings that brains of PD patients have adrastically reduced dopamine level, it was reasoned that they might behelped if the amount of dopamine in the brain were restored to normal.Therefore, among others, L-3,4-hydroxyphenylalanine (L-DOPA), also knowna levodopa, was administered intravenously to patients. L-DOPA, as theimmediate precursor of dopamine is, in contrast to dopamine, capable ofcrossing the blood-brain barrier. After the prolonged administration, aremarkable but brief remission in the patient's symptoms was observedwhich suggested an approach for the treatment of Parkinson's disease.However, this effect is generally associated with long term side effectsand disease progression is not prevented. The reason for the strong sideeffect observed during treatment with levodopa is not known but suggestsa toxic effect of said compound or its metabolites, including dopamine.Furthermore, it has been postulated that L-Dopa and its metabolitedopamine themselves have a toxic effect on neural-tissues and thus,besides alleviating the disease symptoms, may contribute to diseaseprogression. It is important to note that Dopamine does not pass theblood-brain barrier in sufficient quantities, thus only a smallpercentage of L-Dopa reaches the brain after systemic administration.Moreover, L-Dopa is quickly metabolised peripherally, therefore highsystemic L-Dopa doses are required to achieve the clinical effect (3-4gr. L-Dopa/day). In view of the fact that immediate side-effects aredirectly related to L-Dopa peak plasma levels, L-Dopa was, in recentyears, administered in combination therapy with other compounds such asdecarboxylase and COMT (Catechol-amine-O-methyl-transferase) inhibitorsto prevent peripheral metabolisation. To prevent the metabolisation ofdopamine in the brain, MAO (Mono-amine oxidase) inhibitors were alsoused. With these additives it was possible to reduce the daily requireddose of L-Dopa to an average of about 600 mg/day. However, theseadditives were only partially capable of reducing the toxic side effectsof the treatment with levodopa and could not prevent diseaseprogression. Thus, there was an urgent need for an effective treatmentof Parkinson's disease and other levodopa-sensitive neurologicaldiseases which is not associated with or counteracting the side-effectsof L-Dopa therapy including neurotoxicity.

Thus, the technical problem underlying the present invention was toprovide means and methods for treating neurological diseases includingParkinson's disease.

The solution to this technical problem is achieved by providing theembodiments characterized in the claims.

Accordingly, the present invention relates to a pharmaceuticalcomposition comprising Mucuna pruriens seed powder or one or morecomponents, substances, fractions or mixtures of substances obtainedtherefrom and a pharmaceutically acceptable diluent, excipient orcarrier.

Mucuna pruriens is a plant of the family Leguminoseae and is indigenousto tropical countries like India and West Indies. It is an annual,climbing leguminous vine capable of growing to 6 m in length. Thelanceolate leaves are alternate with three large, rhomboid-ovateleaflets. The flowers grow in racemes in 2 or 3 and are white to darkpurple and hang in long racemes. Mucuna pruriens produces clusters ofpods that are curved (4 to 8 cm long) and contain 2 to 6 seeds. Theseeds vary in colour from black, white to mottled. The pods which arethick and leathery are covered with reddish-orange long stiff hairs thatare readily dislodged and can cause intense irritation to the skin.

Since Mucuna pruriens and its use is so widespread that it is consideredcommon fare from China to England, Iran to Spain, Africa to SouthAmerica, it has a variety of common names like Nescafe, Cowage,Velvetbean, Fagiolo Di Rio Negro, Fogarate, Jeukerwt, Juckbohne, Nd,Pien Tou, Pois A Gatter, Pois Gratte, Swagupta, T'Ao Hung King, Kekaragatel or Rarawejah.

Velevetbean, a vigorous annual climbing legume, originally came fromsouthern China and eastern India, where it was at one time widelycultivated as a green vegetable crop. The genus Mucuna, belonging to theFabaceae family, covers perhaps 100 species of annual and perenniallegumes, including the annual velvetbean.

According to Dr. Duke's Phytochemical and Ethnobotanical Databases atphytochemical Database, USDA-ARS-NGRL, Beltsville Agricultural ResearchCenter, Beltsville, Md. Mucuna pruriens contains many diversePhytochemicals like 1-methyl-3-carboxy-6,7-dihydroxy-12,3,4-tetrahydroisoquinolone, 5-hydroxytryptamine,5-methoxy-n,n-dimethyltryptamine-n-oxide, 5-oxyindole-3-alkylamine,6-methoxyharman, Alanine, Arachidic-acid, Arginine, Aspartic-acid,Behenic-acid, Beta-carboline, Beta-sitosterol, Bufotenine, Choline,Cis-12,13-epoxyoctadec-trans-9-cis-acid,Cis-12,13-epoxyoctadec-trans-9-enoic-acid, Cystine, DOPA, Gallic-acid,Glutamic-acid, Glutathione, Glycine, Histidine, L-DOPA, Lecithin,Leucine, Linoleic-acid, Mucunadine, Mucunain, Mucunine, Myristic-acid,N,n-dimethyltryptamine, N,n-dimethyltryptamine-n-oxide, Nicotine,Oleic-acid, Palmitic-acid, Palmitoleic-acid, Phenyalanine, Phosphorus,Proline, Protein, Prurienidine, Prurienine, Saponins, Serine, Serotonin,Stearic-acid, Threonine, Tryptamine, Tyrosine, Valine, Vernolic-acid.Therefore, Mucuna pruriens finds traditionally use in a number ofdiseases and is commonly used as carminative, hypotensive &hypoglycemnic agent. Moreover it is also used as anodyne, antidotal,aphrodisiac, diuretic, nervine, resolvent, rubefacient, and vermifuge;used for anasarca, asthma, cancer, cholera, cough, diarrhea, dogbite,dropsy, dysuria, insanity, mumps, pleuritis, ringworm, snakebite, sores,syphilis, tumors, and worms.

From phytochemistry point of view, the drug contains dimethyltryptaminealkaloids and related alkaloids, lecithin and tannins as well as L-DOPA,a precursor of the neural transmitter dopamine. All these compounds areknown to exist in the seeds of Mucuna pruriens. Therefore, plants likeMucuna pruriens provide a natural source for drugs for Parkinson'sdisease since they contain, among many other phytochemicals, largeamounts of levodopa (L-DOPA).

The term “therapeutically effective” means in an amount sufficient toprevent, treat or ameliorate a disease or the symptoms associated with adisease. The term “obtained” means isolated, extracted or otherwisetaken or gained from the seed. The person skilled in the art knowsvarious techniques for isolating or obtaining compounds from plants,some of which are described below.

The term “pharmaceutically acceptable” means approved by a nationalregulatory agency or by a generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and, oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

The terms “components”, “substances”, “fractions” or “mixtures ofsubstances” all refer to compounds or mixtures of compounds isolatedfrom Mucuna pruriens seeds. The term “isolated” refers to the process ofobtaining or isolating the compound. The isolated component mayinitially be present in a crude extract of the seed, together with manyother components of the seed. Later stages of the extraction processwill yield fractions containing a reduced variety of components. Thismixture of components may have similar physical or chemical properties.Further fractionation, however, will ultimately result in the completeisolation of a single molecular species which is the isolated component.The term “substance” as used herein refers to the isolated or purecomponent. However, methods such as solvent extraction generally resultin a final fraction which contains minute amounts of contaminants.Preferably the substance is 100% pure, less preferably the substance isat least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 80% or at least 70%pure. However, in certain cases also substances with a smaller degree ofpurity can be therapeutically effective. Therefore, the invention alsorefers to substances which are only at least 60%, 55% or even 50% pure.

The pharmaceutical composition, Mucuna pruriens seed powder or extracts,disclosed by the present invention allow long term L-Dopa treatment ofneurological diseases, including Parkinson's Disease, in the absence ofthe short- and long term side effects observed in conventional treatmentapproaches. The term “Mucuna pruriens seed powder” relates to powderprepared from the seeds of Mucuna pruriens. Powder of Mucuna pruriensseeds can be prepared from dried beans which are freed from theircutular hairs by a brushing machine, then milled in a special mill forherbal drugs. The resulting powder is passed through a standard sieveNo. 4 or 5, corresponding to a mesh width of 850 or 355 μm. Thepharmaceutical composition and specific extracts of the presentinvention, not containing L-Dopa, can be applied as such for thetreatment of neurodegenerative diseases in general, or in combinationwith isolated L-Dopa if required.

Conventional L-Dopa therapy requires a gradual increase of the effectivedose over time resulting of progression of disease and/or the neurotoxiceffects of L-Dopa or dopamine with an increase of toxic reactions and,over time, the appearance of dyskinesia, increasing in severity withdose. In clinical experiences with Mucuna prurience seed preparationsthese negative phenomena have not been observed in that for theeffective treatment of Parkinson's, the dose of Mucuna pruriens derivedL-Dopa remained relatively stable over longer periods of time, and inthat dyskinesia, even in patients with pre-existing dyskinesia followinglong term therapy with conventional L-Dopa preparations, appeared to beless in occurrence and severity.

The present invention represents a rationale for this experience.Surprisingly, even in cases of relatively high levels of L-Dopa in bloodfollowing Mucuna pruriens administration, no immediate toxic effectswere encountered normally to be expected with such levels followingadministration of conventional preparations.

The clinical study presented in the Examples of the present inventiondemonstrates that this seed powder formulation of Mucuna prurienscontains a considerable quantity of L-Dopa which is sufficient toconsistently induce a sustained on-period in fluctuating patients withshort duration L-Dopa response. The quality of motor improvementfollowing a single dose challenge was equivalent to that seen withsynthetic LD/DC. Both with the dose of 15 g and 30 g Mucuna pruriens thetime to the beginning of switching “on” and the time to the full“on”-state was significantly shorter than with a pharmaceuticalcomposition containing a combination of synthetic L-Dopa anddecarboxylase inhibitor (LD/DC). The duration of the on-period wassignificantly longer with 30 g Mucuna pruriens than with LD/DC. This wasreflected in the pharmacokinetic profile of L-Dopa plasma levels,showing a significantly steeper slope of increase and earlier T_(max)for Mucuna pruriens and larger total AUCs. For.30 g Mucuna both theearly onset and long duration of effect can in part be explained by theearly and higher peak dose levels and the large AUC after ingestion ofthis dose. Although not significant, the duration of effect of the 15 gMucuna dose tended to be somewhat shorter than with LD/DC. This isreflected in the pharmacokinetic findings in that the L-Dopa levelfollowing this dose decreased below the level of LD/DC after about 1.5hours. In a clinical setting this could therefore be corrected with adose adaptation.

These findings raise the possibility that Mucuna pruriens formulationsmay actually have a faster bioavailability than standard L-Dopapreparations. This is likely to be related to differences in the speedof gastrointestinal absorption of L-Dopa from the duodenum. The mostobvious differences between the Mucuna preparation and the syntheticformulation used in this study was the administration of Mucuna as asuspension as opposed to a capsule, and the addition of a peripheraldecarboxylase inhibitor to the standard L-Dopa preparation.

Decarboxylase inhibitors mainly increase L-Dopa plasma levels byblocking the peripheral degradation of L-Dopa to dopamine, thus allowingmore L-Dopa to cross the blood-brain barrier with an exogenous L-Dopadose reduction of 60-80% (14-17). However, one of the sites fordecarboxylation of oral L-Dopa is the gastric and intestinal mucosa(18), and decarboxylase inhibitors have been reported to enhanceintestinal L-Dopa absorption (19,20), presumably by inhibiting metabolicpathways such as aromatic dehydroxylation in the gut. Other studiesconfirmed that, in the presence of a decarboxylase inhibitor, peakL-Dopa concentrations were higher and were reached more rapidly (14, 21,22).

In the light of these reports, the observations in the underlying studyare surprising. A possible explanation for this may lie in theadministration of Mucuna as a suspension. On the other hand, the L-Dopain the Mucuna seed powder is embedded in organic material. Although itcan not be excluded, it is unlikely that its liberation is so muchquicker than the disintegration of a gelatine capsule in the gastricfluid to be a sufficient explanation for such marked differences inpharmacokinetics. Additives contained in the Mucuna powder formulationmay also have had an impact on absorption: A small amount of ascorbicacid and citric acid, added for chemical stability (23), may potentiallyhave improved intestinal absorption (24). The small amounts actuallyadded to the formulation (0.188 g/unit) do not seem to be a sufficientexplanation however. Furthermore, in a previous clinical trial using aMucuna pruriens formulation not containing such additives the C_(max) ofL-Dopa also appeared to be reached within 1 hour (5). Decarboxylaseinhibitors have been shown to prolong L-Dopa half life (21,22,25).Although the decline of L-Dopa plasma levels on 15 g Mucuna wasslightly, but not significantly, faster than on LD/DC, the L-Dopa levelson 30 g Mucuna declined at the same rate as with LD/DC as shown in theintrapatient analysis (FIG. 2). Therefore it is feasible that inaddition to L-Dopa Mucuna pruriens seed powder contains an absorptionenhancing and potentially also a decarboxylase inhibitor-like factor.

Another surprising finding was that in spite of a much higher L-Dopaexposure on the Mucuna preparations the 3-OMD plasma levels did notincrease accordingly. In fact there was no significant difference in3-OMD levels on 30 g Mucuna (with a 159% higher LD AUC) compared withthe levels on LD/DC. Furthermore, on 15 g Mucuna (LD AUC 31% higher)there was a steady decline of 3-OMD levels reaching a significantdifference with LD/DC (p=0.009 at 240 minutes). If no decarboxylaseinhibitor-like factor is present in Mucuna seed powder this could beexplained by a predominantly metabolisation of L-Dopa bydecarboxylation. Otherwise a doses dependent COMT-like inhibition ofdopamine metabolism by another factor contained in Mucuna pruriens seedpowder could play a role in addition.

Mucuna pruriens seed powder is a natural plant product and therefore bydefinition contains more chemical substances besides L-Dopa. Therefore,and in view of the results of our study, further investigations intofactors potentially promoting gastrointestinal absorption of L-Dopa andaltering its metabolism are warranted.

Tolerability was comparable in all study drugs. All adverse events thatoccurred were mild and short-lasting. Acute side effects of L-Dopa suchas nausea, vomiting, and orthostatic hypotension (dizziness) have beenshown to be correlated with plasma levels (26). In view of thesignificantly higher plasma levels reached on Mucuna than on LD:DC, itis remarkable that side effect profiles were similar in this single dosechallenging study. In fact, but not significantly due to patientnumbers, the number of events on Mucuna was lower compared with LD/DC.

In addition, the results of the AIMS and Goetz scores indicate, that inspite of the significantly higher L-Dopa plasma levels on Mucuna,dyskinesias were not increased on Mucuna compared with LD/DC (27).Furthermore, the Dyskinesia Motor Index even indicates a dose dependentdecrease in the index. This decrease did not reach significance in thissingle dose challenge study, however, due to small patient numbers, butseems to corroborate earlier clinical observations.

Mucuna pruriens contains larger amounts of L-Dopa than any other knownnatural source (28,29). The clinical data summarized above shows thatL-Dopa uptake is considerably increased. It could therefore bepostulated that this Mucuna pruriens may also produce factors to protectvulnerable cells against oxidative damage. In fact, the experimentsperformed in connection with the present invention demonstrate aneuroprotective and neurostimulatory property of Mucuna pruriens.

In order to characterize the active components in Mucuna pruriens andpossibly to identify the factors responsible for the increased uptake ofdopamine and to characterize the neuroprotective properties of Mucunapruriens, a number of in vitro experiments were performed. Initially,the effect of Mucuna pruriens fractions on primary dopaminergic cultureswas tested resulting in the identification of two fractionssignificantly increasing dopamine uptake. In another set of experiments,fractions of Mucuna pruriens extracts were tested with respect for theirpotential to reduce toxic effects of MPP and BSO. The results of theseexperiments allowed the identification of fractions, containing aneuroprotective factor of Mucuna pruriens. In addition, the experimentsdemonstrate that other fractions contain factors that increase cellularuptake of dopamine.

Based on this preliminary single dose, double blinded, double dummychallenging study in patients with Parkinson's Disease and shortduration of L-Dopa response, the Mucuna pruriens formulation seems topossess potential advantages over existing commercially availablesynthetic L-Dopa formulations in that it combines a rapid onset ofaction with a comparable or longer duration of therapeutic responsewithout increasing dyskinesias or acute LD toxicity in spite of muchhigher LD plasma levels. If better tolerability of L-Dopa can beconfirmed in further pre-clinical and larger and longer term clinicalstudies, Mucuna pruriens would provide a wider therapeutic index forL-Dopa treatment for PD patients and thus be a viable alternative tostandard synthetic L-Dopa formulations.

In a preferred embodiment, the components, substances, fractions ormixtures of substances are extracted from Mucuna pruriens seeds.

The term “extraction” refers to the process of obtaining or isolating acompound from Mucuna pruriens seeds. The person skilled in the art knowsof various extraction techniques all of which rely on the physicalproperties of the compounds to be isolated. Extraction, as used herein,relates to the separation of medicinally active portions of Mucunapruriens from the inactive or inert components through the use ofselective solvents. The person skilled in the art knows that the term“extraction” comprises maceration, percolation, digestion, infusion anddecoction. Many extraction methods contain one or more steps ofmechanical treatment of the seed which is usually an initial step thatmay be followed by a filtration, a washing and/or a drying step. Theextraction protocol may be composed of several extraction steps,resulting in the generation of one or more fractions containing variousconcentrations of the therapeutically active compound. However, methodssuch as solvent extraction generally result in a final, fraction whichcontains minute amounts of contaminants. Preferably the substance is100% pure, less preferably the substance is at least 99%, 98%, 97%, 96%,95%, 94%, 93%, 92%, 80% or at least 70% pure. However, in certain casesalso substances with a smaller degree of purity can be therapeuticallyeffective. Therefore, the invention also refers to substances which areonly at least 60%, 55% or even 50% pure. When the extraction protocolcomprises more than one extraction step, the extraction steps may befollowed or preceded by additional steps of pre-treatment. Thesepre-treatments may be important in order to optimise the quality and/orquantity of the next extraction process and may include steps such asmixing, heat-treatment, addition of chemical compounds, filtration,distillation.

In a preferred embodiment, the pharmaceuutical composition comprisebipolar-lipophilic molecules obtained by extraction of Mucuna pruriens.

Solvent extraction relies on differential solvent solubility of the seedcomponents. Polar solvents will tend to extract water-soluble compounds,non-polar solvents will tend to extract lipophilic compounds, whileamphiphilic solvents can extract lipophilic as well as water-solublecompounds. The person skilled in the art knows of various solvents thatcan be used in an extraction process. Preferably the solvent used is asingle solvent selected from the group consisting of water, hexane,acetone, ethanol, chloroform, ethylacetate, diclormethane andpetrolether. However, also preferred are mixtures of two or moresolvents. The solvent may additionally contain compounds such as enzymeinhibitors including phosphatase inhibitors or protease inhibitors,reducing or oxidizing agents or the like including DTT, GSH, ascorbicacid or SO₂-gas, chelating agents including EGTA or EDTA, and mono- ordivalent ions including Mg²⁺, Ca²⁺, Na⁺, Li⁺, Cl⁻, SO₄ ², K⁺, NO₃. Theseadditional compounds present in the solvent or added after extractionmay be important to preserve the physical state of the therapeuticallyactive compound. Another preferred embodiment is extraction in thepresence of gases such as nitrogen or argon which may also be importantfor controlling the oxidative state of the extract.

Physical parameters such as pressure or temperature may play animportant role for extraction procedures since they can have a strongimpact on the state of aggregation of the seed components or of thesolvent. High pressure, e.g., results in liquefaction of carbon dioxideand other gases and may have a strong impact on the result of theextraction process. Carbon dioxide, e.g. is known to be an excellentsolvent at hypercritical conditions. Methods of extracting plantmaterial, including methods of extraction with hypercritical CO₂, areknown in the art (Verdichtete Gase zur Extraktion and Raffinierung, E.Stahl, Springer Verlag, Heidelberg, Berlin, 1986). Accordingly, theextraction process of the present invention may be performed at lowpressure, i.e. at low pressure: <200 bar, intermediate pressure: 200-300bar, high pressure: >300 bar. Similarly, the temperature during theextraction process can be important for the yield of the extractionprocess. Accordingly the present invention can be performed at lowtemperature, i.e. between 0° C. and 10° C., at an intermediatetemperature, i.e. between 10° C. and 40° C., preferably 20° C., 25° C.,30° C. or 37° C., or at high temperature, i.e. between 40° C. and 100°C.

Accordingly, the present invention relates in a preferred embodiment toa method for the preparation of extracts or extract fractions of Mucunapruriens, comprising extracting the seed of Mucuna pruriens with CO₂ ormixtures from CO₂ and butane, propane or other gases under supercriticalconditions or different pressures and temperatures, to obtainpurification and selection of substances or fractionation of Mucunapruriens extracts.

In a preferred embodiment of the present invention, Mucuna pruriensseeds or seed powder is extracted twice with acetone. The remainingmaterial is further extracted with n-propanol.

In another preferred embodiment of the present invention, Mucunapruriens seeds or seed powder is extracted at least one time with a 1:1mixture of water and ethanol. The extraction process can be performed inthe presence or absence of ascorbic acid. In another preferredembodiment of the present invention, Mucuna pruriens seeds or seedpowder is initially extracted by water, the resulting extract is furtherfractionated by ethanol precipitation.

In a more preferred embodiment, the components, substances, fractions ormixtures of substances are extracted from Mucuna pruriens seeds by usingbipolar-lipophilic solvent molecules such as acetone, DMSO ordimethylformamide which extract liphophilic and a great part of polar ofhydrophilic substances from the plant material. These solvents aresometimes also designated simply as bipolar. Other extraction methodsare staggered procedures starting with organic solvents followed bypolar solvents or vice versa. Very often also water-alcohol,alcohol-acetone or acetone-hexane solvent mixtures are used to extractlipophilic and hydrophilic constitutents in one extraction operation.Preferably the bipolar-lipophilic solvent is selected from the groupconsisting of acetone, DMSO or dimethylformamide. However, this list isnon-limiting and the person skilled in the art knows of many otherbipolar-lipophilic molecules which might be used in the extractionprocess.

In another preferred embodiment, the pharmaceutical composition isformulated as an infusion, an injection solution, a gelatin-capsule, atablet or a controlled release tablet. Many delivery systems are knownto the person skilled in the art and can be used to administer acompound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules. Methods of introduction include but, arenot limited to intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal, epidural, and oral routes. The compounds orcompositions may be administered by any convenient route, for example byinfusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa,etc.) and may be administered together with other biologically activeagents. Administration can be systemic or local. In addition, it may bedesirable to introduce the pharmaceutical compounds or compositions ofthe invention into the central nervous system by any suitable route,including intraventricular and intrathecal injection; intraventricularinjection may be facilitated by an intraventricular catheter, forexample, attached to a reservoir, such as an Ommaya reservoir. Pulmonaryadministration can also be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent, it may bedesirable to administer the pharmaceutical compounds or compositions ofthe invention locally to the area in need of treatment. The compound orcomposition can be delivered in a vesicle, in particular a liposome (seeLanger, Science 249:1527-1533 (1990); Treat et al., in Liposomes in theTherapy of Infectious Diseases and Cancer, Lopez-Berestein and Fidler(eds.), Liss, N.Y., pp. 353-365 (1989); Lopez-Berestein, ibid., pp.317-327; see: generally ibid.). The compound or composition can bedelivered in a controlled release system, including the use of a pump(see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987);Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.321:574 (1989)). The above-mentioned controlled release system can beplaced in proximity of the therapeutic target, i.e., the brain, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138(1984)).

In another preferred embodiment, the present invention's pharmaceuticalcomposition comprises (a) a neurostimulatory extract of Mucuna pruriensselected from the group consisting of M-PL0100, M-EL100, M-BL0100 andLAT543-0 or (b) a neuroprotective extract of Mucuna pruriens selectedfrom the group consisting of M-W-EL1299, M-W0100, MWEL0700 and M-ML0100.One or more of the extracts may be combined in the same pharmaceuticalcomposition.

The term “M-PL0100” refers to an extract obtainable, from Mucunapruriens by shaking 10 g of the pulverised seeds of M. pruriens,preferably for 18 hours at room temperature, in n-propanol (50 ml) andby filtering subsequently. The extraction of the residue may be followedby one, two or more additional steps of shaking with n-propanol andfiltering. The filtrates may be pooled together and the solventdistilled off to get an oily mass. Of course, larger or smallerquantities of M-PL0100 can be prepared, simply by reducing or increasingthe amount of seeds and solvent.

The term “M-EL100” refers to an extract obtainable from Mucuna pruriensby shaking 20 g of pulverised seeds of M. pruriens, preferably for 18hours, in EtOH (100 ml). After filtration, the residue is shaken,preferably for 18 hours, and filtered. The process may be repeated two,three, four or up to ten times. Subsequently, the filtrates may beconcentrated and pooled together to get the extract. Of course, largeror smaller quantities of M-EL100 can be prepared, simply by reducing orincreasing the amount of seeds and solvent.

The term “M-BL0100” relates to an extract obtainable from Mucunapruriens by shaking 10 g of pulverised seeds of M. pruriens, preferablyfor 18 hours at room temperature in n-butanol (50 ml). Preferably a stepof filtering follows. Subsequently, the residue may be extracted two ormore times with n-butanol, preferably followed by a step of filtering.The filtrates may be pooled together and the solvent distilled off. Theoily extract is thus obtained. Of course, larger or smaller quantitiesof M-BL0100 can be prepared, simply by reducing or increasing the amountof seeds and solvent.

The term “LAT00270543” is defined as “LAT543” and the term“LAT00270543-0” is defined as “LAT543-0”. Both LAT543 and LAT543-0 arepolysaccharide fractions. They, may be obtained from a water extractafter a preceded hexane, dichlormethane and methanol soxhlet extraction.The organic solvent extract is discarded. The water extraction may becarried out for 1 hour at 80° C. After cooling to room temperature, 1 gNaN₃ may be added. The solution may be centrifuged for 30 min at 8000Upm and washed twice with distilled water. The solution may be dialysedat 4° C. in a VIKING dialysis tubing 36/32, diameter 27 mm for 96 hourswith 12 times water exchange. The dialysed water fraction may becentrifuged and freeze-dried. The term LAT543 relates to a precipitateobtainable from 3 g of the brownish residue of the hot water dissolvedin 300 ml distilled water and adding under stirring at4° C. 300 mlethanol (1:1 water:ethanol) drop by drop. The precipitation may becentrifuged, dissolved in a small amount of distilled water andfreeze-dried whereas the term LAT₅₄₃-0 relates to a precipitateobtainable from the supernatans of LAT-543 precipitation treated in thesame way as said above with 900 ml ethanol (1:4 water:ethanol) to getthe polysaccharide precipitation of LAT543-0. Precipitates of bothLAT543 and LAT543-0 may be freeze-dried and dissolved in water in variesconcentrations for testing.

Yield g g Non dialyzable hot water extract 16.7 3.34 1:1 ethanolprecipitation 2.01 0.40 1:4 ethanol precipitation 0.04 0.01 Total amountof polysaccharides 2.05 0.41

The sugar content of LAT543 and LAT543-0 may be quantified according toBLAKENEY et. al. (1983). The uronic acid content of LAT543 and LAT543-0may be quantified according to BLUMENKRANZ and ASBOE-HANSEN (1973). Thenitrogen content as albumine of LAT543 and LAT543-0 may be quantifiedaccording to LOWRY (1951).

The term “M-W-EL1299” relates to an extract obtainable from Mucunapruriens by shaking 200 g pulverised seed material of Mucana pruriens,preferably at room temperature, in 200 ml n-hexane, preferably for 18hours. After filtration, the material may be washed with 100 ml n-hexaneand filtered. The filtrates may be collected and the solvent distilledoff to obtain a yellowish oil. The residue of the n-hexane extraction issubsequently shaken, preferably for 18 hours, at room temperature, inacetone (200 ml), preferably followed by a step of filtration. Theresidue may be extracted one or more times with 200 ml acetone byfurther shaking, preferably for 18 hours, followed by a step offiltration. Subsequently, the residue may be washed with acetone (100ml) and filtered. After pooling, the filtrates may be evaporated bydistillation, preferably under reduced pressure, yielding a yellowishmass. The residue (obtained from the above extractions, ca. 96 g) isshaken, preferably for 18 hours at room temperature, in 500 ml of amixture of water-EtOH, 1:1 with 0.5% ascorbic acid. The solvent may befiltered and concentrated, preferably under reduced pressure at atemperature of 35° C. The extraction procedure may repeated one, two,three, four or more times. After concentration, the filtrates may becollected and the solvent removed under vacuum to get a solid mass. Ofcourse, larger or smaller quantities of M-W-EL1299 can be prepared,simply by reducing or increasing the amount of seeds and solvent.

The term “M-W0100” relates to an extract obtainable from Mucuna pruriensby shaking the M-EL0100 extract (vide supra), preferably for 18 hours atroom temperature in de-mineralized water, followed by a step offiltration. The extraction may be repeated one, two, three, four or moretimes. Subsequently, the filtrates may be pooled together and waterdistilled off, after passing SO₂ to prevent the oxidation of L-DOPA. Ofcourse, larger or smaller quantities of M-W0100 can be prepared, simplyby reducing or increasing the amount of input material (M-EL0100fraction) and solvent.

The term “MWEL0700” relates to an extract obtainable from Mucunapruriens by extracting Mucuna pruriens with a 1:1 mixture ofwater:ethanol, preferably without ascorbic acid addition, by shaking 100g powder with 500 ml of this solvent mixture, preferably for 18 hrs atroom temperature. The solvent may be evaporated at reduced temperatureto dryness. Of course, larger or smaller quantities of “MWEL0700” can beprepared, simply by reducing or increasing the amount of Mucuna prurienspowder and solvent.

The term “M-ML0100” relates to an extract obtainable from Mucunapruriens by shaking 10 g of pulverized steeds of M. pruriens, preferablyfor 18 hours at room temperature, in methanol (50 ml), followed by astep of filtration. The extraction of the residue is followed by two ormore steps of extraction with methanol followed by a filtration step.The filtrates may be pooled together and the solvent distilled off toyield a semi-solid mass. Of course, larger or smaller quantities of“M-ML0100” can be prepared, simply by reducing or increasing the amountof Mucuna pruriens powder and solvent.

In certain cases it may be preferable to reduce or increase theincubation time with the solvent. Accordingly, the incubation time maybe less than 18 hours, i.e. 17, 16, 15, 14, 13, 12, 11, 10 hours or morethan 18 hours, i.e. 19, 20, 21, 22, 23 or 24 hours. When performing theextraction under pressure, substantially less time may be sufficient toprepare the extract. Preferably, the extraction is performed at roomtemperature, however, in certain cases it may be preferable to reduce orincrease the temperature in order to improve the amount or quality ofthe extract. Accordingly, also preferred are temperatures such as about0° C., 1° C., 2° C., 3° C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C.,10° C., 11° C., 12° C., 13° C., 14° C., 15° C., 16° C., 17° C., 18° C.,19° C., 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C.,28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C.,37° C., 38° C., 39° C. or 40° C., wherein these temperatures may vary by±0.5° C.

The present invention also relates to the use of Mucuna pruriens seedsor of one or more components, substances, fractions or mixtures ofsubstances obtained or extracted from Mucuna pruriens for thepreparation of a pharmaceutical composition (a) for inhibiting L-Dopaand/or dopamine metabolism; (b) for improved L-Dopa absorption,resulting in an earlier onset of L-Dopa efficacy; and (c) for a longerduration of L-Dopa efficacy.

Moreover, the present invention also provides the use of Mucuna pruriensseeds or one or more components, fractions or mixtures of substancesobtained or extracted from Mucuna pruriens for the preparation of apharmaceutical composition for neuroprotection or neurostimulation.

The term “neuroprotection” means protection of nerve tissues.Furthermore, neuroprotection is the protection of nerve cells and theirfunction against endogene or exogene physical or chemical factorsnegatively influencing the metabolism, survival or function of nervecells such as for example heat application, Roentgen radiation,ischaemia, neurotoxins, oxidants, intoxications including heavy metals,infections and sequelae of vaccinations, systemic metabolic diseases anddisturbances in endocrine or electrolyte homeostasis.

The term “neurostimulation” means stimulation of nerve tissues.Moreover, neurostimulation is the improvement, enhancement orrestoration of function of nerve cell tissues, both centrally andperipherally, through stimulation of cellular growth or nerve cellactivity, both with respect to signal conduction and transduction, byphysical means such as for example electro-stimulation orchemical-pharmacological means. The neuroprotective effect of Mucunapruriens is registered by in vitro measurements of the survival andgrowth rate of mesencephalic or motor-neurons after pretreatment withMucuna extract and exposure to a damaging (oxidative, toxic) stressagents.

The present invention also provides the use of one or more Mucunapruriens components, fractions or mixtures of substances obtained orextracted from Mucuna pruriens for the preparation of a pharmaceuticalcomposition for preventing, alleviating or treating neurologicaldiseases. The term “neurological disease” includes diseases such asParkinson disease (PD), Alzheimer's disease (AD), amyotrophic lateralsclerosis (aALS), motoneuron disease.

A preferred embodiment of the invention the use relates to neurologicaldegenerative diseases and comprises a large number of diseases known tothe person skilled in the art. According to the present invention,neurodegenerative or neurological degenerative diseases fall into one ofthe following groups A to D:

-   A: Degenerative and Heredodegenerative Diseases (nerve tissue    atrophy is primair). This group includes, but is not limited to:    Parkinson's disease, Chorea Huntington, Hallervorder-Spatz disease,    Alzheimer's disease, senile dementia, Creutzfeldt-Jakob disease,    artheriosclerotic dementia, cerebral thrombangitis obliterans.-   B: Metabolic and nutritional Disorders (secondair to systemic    disorder). This group includes, but is not limited to: disturbances    in lipoid metabolism (a.o. Gaucher, Niemann-Pick, Tay-Sachs, Hurler,    Refsum), Leukodystrophien, disturbances in aminoacid, carbohydrate    metabolism, hepatolenticular degeneration, etc. deficiencies such as    Vitamin B12 deficiency or folic acid deficiency.-   C: Systemic diseases, endocrine disturbances and autoimmune    reactions afflicting the nervous system: This group includes, but is    not limited to: hypothyreose, hypo- and hyperparathyreoidismus,    Collageen diseases, systemic lupus erythematodes, sarcoidosis,    leukoencephalopathy, demyelisation.-   D: Nervous tissue damage by various endogenic and exogenic factors:    This group includes, but is not limited to: ischemiam, trauma's,    physical noxen, intoxications with metals (mercury, lead, aluminum,    etc) and neurotoxins, alcohol abuse, sequelae of infection and    vaccination.

Neurological degenerative diseases, as used herein, that may beprevented, treated or alleviated are diseases characterized by theformation of nervous system lesions. These lesions include but are notlimited to, the following lesions of either the central (includingspinal cord, brain) or peripheral nervous systems: (1) ischemic lesions,in which a lack of oxygen in a portion of the nervous system results inneuronal injury or death, including cerebral infarction or ischemia, orspinal cord infarction or ischemia; (2) traumatic lesions, includinglesions caused by physical injury or associated with surgery, forexample, lesions which sever a portion of the nervous system, orcompression injuries; (3) malignant lesions, in which a portion of thenervous system is destroyed or injured by malignant tissue which iseither a nervous system associated malignancy or a malignancy derivedfrom non-nervous system tissue; (4) infectious lesions, in which aportion of the nervous system is destroyed or injured as a result ofinfection, for example, by an abscess or associated with infection byhuman immunodeficiency virus, herpes zoster, or herpes simplex virus orwith Lyme disease, tuberculosis, or syphilis; (5) degenerative lesions,in which a portion of the nervous system is destroyed or injured as aresult of a degenerative process including but not limited to,degeneration associated with Parkinson's disease, Alzheimer's disease,Huntington's chorea, or amyotrophic lateral sclerosis (ALS); (6) lesionsassociated with nutritional diseases or disorders, in which a portion ofthe nervous system is destroyed or injured by a nutritional disorder ordisorder of metabolism including, but not limited to, vitamin B12deficiency, folic acid deficiency, Wernicke disease, tobacco-alcoholamblyopia, Marchiafava-Bignami disease (primary degeneration of thecorpus callosum), and alcoholic cerebellar degeneration; (7)neurological lesions associated with systemic diseases including, butnot limited to, diabetes (diabetic neuropathy, Bell's palsy), systemiclupus erythematosus, carcinoma, or sarcoidosis; (8) lesions caused bytoxic substances including alcohol, lead, or particular neurotoxins; and(9) demyelinated lesions in which a portion of the nervous system isdestroyed or injured by a demyelinating disease including, but notlimited to, multiple sclerosis, human immunodeficiency virus-associatedmyelopathy, transverse myelopathy or various etiologies, progressivemultifocal leukoencephalopathy, and central pontine myelinolysis.Neurological degenerative disease associated with the formation oflesions and/or behavioral disorders include, but are not limited to,Alzheimers Disease, Parkinsons Disease, Huntingtons Disease, TouretteSyndrome, schizophrenia, mania, dementia, paranoia, obsessive compulsivedisorder, panic disorder, learning disabilities, ALS, psychoses, autism,and altered behaviors, including disorders in feeding, sleep patterns,balance, and perception.

In a more preferred embodiment the neurological degenerative disease isselected from the group of degenerative and heredodegenerative diseasesincluding Parkinson's disease, Chorea Huntington, Hallervorder-Spatzdisease, Alzheimer's disease, senile dementia, Creutzfeldt-Jakobdisease, artheriosclerotic dementia, cerebral thrombangitis obliteransor other diseases, according to any one of the diseases mentioned undergroup A to D of neurodegenerative diseases, which can be caused byexogenic or endogenic factors. The term “endogenic factor” meansoriginating from within an organism, the term “exogenic factor” meansoriginating from outside the organism.

Yet another more preferred embodiment relates to a neurologicaldegenerative disease which is Parkinson's disease. In a particularlypreferred embodiment of the present invention, Parkinson's disease istreated by preventing acute or chronic L-Dopa toxicity. As the presentinvention discloses that in patients treated with Mucuna pruriens muchhigher levels of L-Dopa are tolerated without the induction of toxicside-effects, Mucuna pruriens and its extracts may be used forsuppressing toxic side effects. Particularly preferred are thosefractions or extracts of Mucuna pruriens which show neuroprotective orneurostimulatory activity.

Still another preferred embodiment relates to the use of Mucuna pruriensseed powder or one or more Mucuna pruriens components, fractions ormixtures of substances obtained or extracted from Mucuna pruriens forthe preparation of a pharmaceutical composition for preventing,alleviating or treating a neurological degenerative disease, whereinsaid components, fractions or mixtures of substances obtained orextracted from Mucuna pruriens can be or contain any chemical entitycontained in Mucuna pruriens. However, preferably said components,fractions or mixtures of substances are selected from the groupconsisting of alkaloids, proteins, peptides, polysaccharides,glycosides, glycoproteins, sterols, phytochemicals like1-methyl-3-carboxy-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinolone,5-hydroxytryptamine, 5-methoxy-n,n-dimethyltryptamine-n-oxide,5-oxyindole-3-alkylamine, 6methoxyharman, Alanine, Arachidic-acid,Arginine, Aspartic-acid, Behenic-acid, Beta-carboline, Beta-sitosterol,Bufotenine, Choline, Cis-12,13-epoxyoctadec-trans-9-cis-acid,Cis-12,13-epoxyoctadec-trans-9-enoic-acid, Cystine, DOPA, Gallic-acid,Glutamic-acid, Glutathione, Glycine, Histidine, L-DOPA, Lecithin,Leucine, Linoleic-acid, Mucunadine, Mucunain, Mucunine, Myristic-acid,N,n-dimethyltryptamine, N,n-dimethyltryptamine-n-oxide, Nicotine,Oleic-acid, Palmitic-acid, Palmitoleic-acid, Phenyalanine, Phosphorus,Proline, Protein, Prurienidine, Prurienine, Saponins, Serine, Serotonin,Stearic-acid, Threonine, Tryptamine, Tyrosine, Valine, Vernolic-acid orphosphatides such as phosphatidylcholin, phosphatidylethanolamine,phosphatidylserin, phosphatidylinositol. Preferred alkaloids includeL-3-Carboxy-6,7-dhihydroxy-1,2,3,4-tetrahydroisoquinoline, L-3-Carboxy7,8-dhihydroxy-1,1-dimethyl, 1,2,3,4tetra-hydroisoquinoline,L-3-Carboxy-6,7-dihydroxy-1,1-dimethyl1,2,3,4-tetra-hydroisoquinoline,L3-Carboxy-6,7-dihydroxy-1 β-methyl-1,2,3,4-tetraisoquinoline and1-methy-3-carbox-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline. Morepreferably said components, fractions or mixtures of substances containL-Dopa and one or more components contained in Mucuna pruriens. Anotherpreferred embodiment of the invention relates the use of components,substances, fractions or mixtures of substances isolated form Mucunapruriens which do not contain a pharmaceutically effective amount oronly traces of L-Dopa.

Another preferred embodiment of the invention relates to the use ofalcohols and/or mixtures thereof, used for the extraction of Mucunapruriens components, fractions or mixtures of substances, wherein thealcohol is selected from the group consisting of hexanol, ethanol,methanol, isopropanol, n-butanol and propanol.

A further preferred embodiment relates to the use of organic solventsand/or mixtures thereof, used for the extraction of Mucuna prurienscomponents, fractions or mixtures of substances, wherein the organicsolvent is selected from the group consisting of chloroform, CO₂,hypercritical CO₂, ether, DMSO, hexane, ethylacetate, dichlormethane andacetone.

Yet another preferred embodiment relates to the use of polar solventsand/or mixtures thereof, used for the extraction of Mucuna prurienscomponents, fractions or mixtures of substances, wherein the polarsolvent is selected from the group consisting of water, ethanol,methanol, propanol and isopropanol.

A more preferred embodiment relates to the use of a 1:1 mixture of waterand ethanol containing 0.5% ascorbic acid [% w/w]. Preferably this stepis preceded by an initial extraction step with n-hexane followed by anextraction step with acetone.

However, the invention also relates to the use of mixtures, wherein theethanol concentration is preferably between 10% and 75%, more preferredbetween 20% and 65%, even more preferred between 30% and 60% and mostpreferred 50%. Preferably said mixture contains between 0.01% and 2%ascorbic acid, more preferred between 0.1% and 1.2%, even more preferredbetween 0.3% and 0.9% and most preferred 0.5% ascorbic acid. However, insome cases it may be advantageous to add no ascorbic acid to theextraction solution.

Another preferred embodiment relates to the use of one or more solventsor mixtures of the solvents for the extraction process. According tothis embodiment, two or more solvents selected from the group ofalcohols, organic solvents and polar solvents are used for theextraction process.

Still another embodiment relates to fractionated extraction.

In a preferred embodiment of the present invention's use, the extract ofMucuna pruriens is (a) a neurostimulatory extract of Mucuna pruriensselected from the group consisting of M-PL0100, M-EL100, M-BL0100 andLAT543-0 or (b) a neuroprotective extract of Mucuna pruriens selectedfrom the group consisting of M-W-EL1299, M-W0100, MWEL0700 and M-ML0100.One or more of the extracts may be combined in the same pharmaceuticalcomposition.

In another preferred embodiment, the present invention's pharmaceuticalcomposition comprises (a) a neurostimulatory extract of Mucuna pruriensselected from the group consisting of M-PL0100, M-EL100,M-BL0100 andLAT543-0 or (b), a neuroprotective extract of Mucuna pruriens selectedfrom the group consisting of M-W-EL1299, M-W0100, MWEL0700 and M-ML0100.One or more of the extracts may be combined in the same pharmaceuticalcomposition.

The present invention also provides a method of preparing extracts orextract-fractions of Mucuna pruriens comprising (a) extracting seeds ofMucuna pruriens with n-hexane to provide a first extract solution; (b)filtering the first extract solution; (c) extracting the filterretentate of (b) with acetone to provide a second extract solution; (d)filtering the second extract solution; (e) extracting the filterretentate of (d) with a 1:1 mixture of water and ethanol containing 0.5%ascorbic acid to provide third extract solution; (f) filtering the thirdextract solution; (g) repeating at least four times the extractionprocedure of (e) with the retentate obtained by (f); and (h)concentrating the pooled extract solutions. However, the invention alsorelates to methods comprising additional filtration, washing orextraction steps. The term “filtration” means filtration by passingthrough a filter. The term “washing” means to wash the filtered residueon a filter with the mentioned solvent. The term “concentrating” meansevaporation at low temperature (40-75° C.) and at normal or underreduced pressure. Water extract could alternatively be freeze-dried.

Furthermore, the invention also relates to a method wherein hexane inthe initial step and/or acetone in the second extraction step isreplaced by at least one other organic solvent as defined above.Furthermore, the invention also relates to a method wherein the ethanolcontent and/or the content of ascorbic acid are modified. In particular,the ethanol concentration may be modified to any concentration between10% and 75%, more preferred between 20% and 65%, even more preferredbetween 30% and 60% and most preferred between 45% and 55%. Preferablysaid mixture contains between 0.01% and 2% ascorbic acid, more preferredbetween 0.1% and 1.2%, even more preferred between 0.3% and 0.9% andmost preferred between 0.45% and 5.5% ascorbic acid. However, in somecases it may be advantageous to add no ascorbic acid to the extractionsolution. Furthermore, this method of the invention allows replacementof ethanol with other alcoholic compounds selected from the groupconsisting of propanol, isopropanol and methanol. The method forpreparing extracts or extract-fractions of Mucuna pruriens comprisesrepeating the extraction procedure of step (e) on the retenate of step(f). Nevertheless, in some cases it may be advantageous to eliminatethis step from the method or to repeat this step at least once, at leasttwice, at least three times, at least four times, at least five times orup to ten times.

The present invention also provides a method of preparing extracts orextract-fractions of Mucuna pruriens comprising (a) extracting seeds ofMucuna pruriens with an alcohol, wherein the alcohol is (i) methanol,ethanol and/or propanol to provide a first extract solution; (b)filtering the first extract solution; (c) repeating at least two timesthe extraction procedure of (a) with the retentate obtained by (b); andconcentrating the pooled extract solutions. Preferably the first extractsolution consists of 10-100% alcohol, more preferably of 30-100%alcohol, even more preferably of 70-100% alcohol, still more preferablyof 90-100% alcohol and most preferably of 99-100% alcohol. This methodfor preparing extracts or extract-fractions of Mucuna pruriens comprisesrepeating the extraction procedure of step (a) with the retenate of step(b). In some cases it may be advantageous to eliminate this step fromthe method or to repeat this step at least once, at least twice, atleast three times, at least four times, at least five times or up to tentimes. Furthermore, in some cases it may be advantageous to add organicsolvents such as e.g. Dimethyl Sulfoxide (DMSO) or water.

In a preferred embodiment the method further comprises solubilizihngsaid extract or extract-fractions of Mucuna pruriens in a solventcomprising DMSO and/or distilled water. Resolubilization may besupported by heat treatment of the extract in the presence of thesolubilizing agent, appropriate conditions can easily established by theperson skilled in the art.

The invention also relates to a method for the preparation of extractsof extract fractions of Mucuna pruriens, comprising extracting the seedof Mucuna pruriens with CO₂ or mixtures from CO₂ and butane, propane orother gases under supercritical conditions or different pressures andtemperatures, to obtain purification and selection of substances orfractionation of Mucuna pruriens extracts.

The present invention also provides the use of Mucuna pruriens seeds orseed powder, as well as extracts or extract fractions of Mucunapruriens, obtainable by the methods of the invention, for thepreparation of a pharmaceutical composition for treating neuronaldiseases. The term “obtainable” or “obtained” means produced, isolatedor extracted by any of the methods of the invention.

A preferred embodiment of the invention relates to the use of extractsor extract-fractions or of extracted components, substances or mixturesof substances wherein Mucuna pruriens is used in comminuted form, asgranules, powder, precipitate, fraction, extract, dried extract and/orexudates, preferably as extract.

Another preferred embodiment of the invention relates to the use ofMucuna pruriens seeds or seed powder or extracts of Mucuna pruriens orextract-fractions or of extracted components, substances or mixtures ofsubstances, wherein said Mucuna pruriens components, substances,fractions or mixtures of substances obtained therefrom are used incombination with one or more other active agents. The term “activeagent” as used herein relates to therapeutic agents or productsclinically used or to be used in future, such as components contained inamino acid fractions with or without L-DOPA, isoquinoline alcaloidfractions, polysaccharide or glycoprotein fractions, phosphatides, fattyacid fractions for the treatment of neurological diseases in which acombination of Mucuna pruriens components, substances, fractions ormixtures of substances with the active agent could be of clinicalbenefit.

Mucuna pruriens components, substances, fractions or mixtures ofsubstances will be formulated and dosed in a fashion consistent withgood medical practice, taking into account the clinical condition of theindividual patient (especially the side effects of treatment with saidMucuna pruriens extract alone), the site of delivery, the method ofadministration, the scheduling of administration, and other factorsknown to practitioners. The “effective amount” for purposes herein isthus determined by such considerations.

Pharmaceutical compositions containing the extracts of the invention areadministered orally, rectally, parenterally, intracistemally,intravaginally, intraperitoneally, topically (as by powders, ointments,gels, drops or transdermal patch), bucally, or as an oral or nasalspray. “Pharmaceutically acceptable carrier” refers to a non-toxicsolid, semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. The term “parenteral” as used hereinrefers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

The extract is also suitably administered by sustained-release systems.Suitable examples of sustained-release compositions includesemi-permeable polymer matrices in the form of shaped articles, e.g.,films, or mirocapsules. Sustained-release matrices include polylactides(U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., Biopolymers, 22:547-556 (1983)),poly (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater.Res. 15:167-277 (1981), and Langer, Chem. Tech., 12:98-105, (1982)),ethylene vinyl acetate (R. Langer et al.) or poly-D-(−)-3-hydroxybutyricacid (EP 133,988). Sustained-release compositions also includeliposomally entrapped extracts. Liposomes containing the Mucuna pruriensextract are prepared by methods known by the person skilled in the art.Ordinarily, the liposomes are of the small (about 200-800 Angstroms)unilamellar type in which the lipid content is greater than about 30mol. percent cholesterol, the selected proportion being adjusted for theoptimal therapy.

For parenteral administration, in one embodiment, the extract isformulated generally by mixing it at the desired degree of purity, in aunit dosage injectable form (solution, suspension, or emulsion), with apharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example., the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to the therapeutical effectiveness of Mucunapruriens extracts. Generally, the formulations are prepared bycontacting the Mucuna pruriens extract uniformly and intimately withliquid carriers or finely divided solid carriers or both. Then, ifnecessary, the product is shaped into the desired formulation.Preferably the carrier is a parenteral carrier, more preferably asolution that is isotonic with the blood of the recipient. Examples ofsuch carrier vehicles include water, saline, Ringer's solution, anddextrose solution. Non-aqueous vehicles such as fixed oils and ethyloleate may also be useful herein, as well as liposomes.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, manose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

The Mucuna pruriens extract is typically formulated in such vehicles ata concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml,at a pH of about 3 to 8.

Any Mucuna pruriens extract to be used for therapeutic administrationcan be sterile. Sterility is readily accomplished by filtration throughsterile filtration membranes (e.g., 0.2 micron membranes). TherapeuticMucuna pruriens compositions generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.

Mucuna pruriens ordinarily will be stored in unit or multi-dosecontainers, for example, sealed ampoules or vials, as an aqueoussolution or as a lyophilized formulation for reconstitution. Theinfusion solution is prepared by reconstituting the lyophilized Mucunapruriens extract using bacteriostatic Water-for-Injection.

Another preferred embodiment of the invention relates to the use ofextracts or extract-fractions or of extracted components, substances ormixtures of substances wherein the Mucuna pruriens components,substances, fractions or mixtures of substances are formulated asinfusion solution, injection solution, for oral forms of application, asa therapeutic pack, a granulate, a food supplement or in form ofclysters.

Yet another preferred embodiment of the invention relates to the use ofextracts or extract-fractions or of extracted components, substances ormixtures of substances in oral, topical and/or parenteral applications.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with Mucuna pruriens components, substances,fractions or mixtures of substances or the pharmaceutical compositionsof the invention. Associated with such container(s) can be a notice inthe form prescribed by a national health authority regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration. In addition, the Mucuna pruriens extracts ofthe present invention may be employed in conjunction with othertherapeutic compounds.

The invention also relates to a method of treatment of an individual inneed of a neuroprotective or neurostimulatory activity comprisingadministering to such an individual a pharmaceutical compositioncomprising an amount of Mucuna pruriens seeds or of one or morecomponents, substances, fractions or mixtures of substances obtained orextracted from Mucuna pruriens which is effective in providing therequired neuroprotective or neurostimulatory activity in such anindividual. For example, a Parkinson disease patient treated with L-Dopatypically develops acute toxic side effects and side effects followinglong term use such as such as dyskinesia. By administering to anindividual a pharmaceutical composition comprising Mucuna pruriens seedsor an extract thereof, said toxic side effects are less severe, absentor are deferred. The pharmaceutical composition containing Mucunapruriens components may be administered as a formulation of whole seedpowder, an extract thereof, or may be administtered as extract fractionsin combination with L-Dopa or separately.

The present invention also relates to a method of treatment of anindividual in need of a wider therapeutic window in L-Dopa therapy andto prevent an early need for combination therapy with, for example, adecarboxylase inhibitor, a decarboxylase, COMT, or MAO inhibitor, orother anti-Parkinson drugs such as for example amantadine, pergolide,ropinirole, cabergoline, pramipexole, said method comprisingadministering to such an individual a pharmaceutical compositioncomprising an amount of Mucuna pruriens seeds or of one or morecomponents, substances, fractions or mixtures of substances obtained orextracted from Mucuna pruriens which is effective in providing therequired wider therapeutic window in such an individual. A widertherapeutic window may allow to treat patients much longer with L-Dopaand with higher L-Dopa doses without the need for combination therapyand the resulting negative side-effects thereof.

The present invention also relates to a method of treatment of anindividual, preferably a Parkinson's disease patient, in need of apharmaceutical composition capable of suppressing the toxic acute and/orchronic side effects of L-Dopa, said method comprising administering tosuch an individual a pharmaceutical composition comprising an amount ofMucuna pruriens seeds or of one or more components, substances,fractions or mixtures of substances obtained or extracted from Mucunapruriens which is effective in suppressing the toxic acute and/orchronic side effects of L-Dopa in such an individual.

The present invention also relates to a method of treatment of anindividual, preferably a Parkinson's disease patient, in need of anearly onset of L-Dopa activity, said method comprising administering tosuch an individual a pharmaceutical composition comprising an amount ofMucuna pruriens seeds or of one or more components, substances,fractions or mixtures of substances obtained or extracted from Mucunapruriens which is effective in providing an early onset of L-Dopaactivity in such an individual. Said extract of Mucuna pruriens may bethe source of L-Dopa, however, exogenous L-Dopa may be added to thepharmaceutical composition. This method is based on the surprisingclinical observation that patients treated with Mucuna pruriens show anincreased absorption of L-Dopa and early onset of L-Dopa activity.

Moreover, the present invention also relates to a method of treatment ofan individual, preferably a Parkinson's disease patient, in need ofsustained L-Dopa plasma levels without a need for combination therapy toinhibit peripheral L-Dopa conversion, said method comprisingadministering to such an individual a pharmaceutical compositioncomprising an amount of Mucuna pruriens seeds or of one or morecomponents, substances, fractions or mixtures of substances obtained orextracted from Mucuna pruriens which is effective in providing thesustained L-Dopa plasma level in such an individual.

Finally, the present invention relates to a method of treatment of anindividual, preferably a Parkinson's disease patient, in need of aninhibition of L-Dopa and/or dopamine metabolism, said method-comprisingadministering to such an individual a pharmaceutical compositioncomprising an amount of Mucuna pruriens seeds or of one or morecomponents, substances, fractions or mixtures of substances obtained orextracted from Mucuna pruriens which is effective inhibiting L-Dopaand/or dopamine metabolism in such an individual.

Typically, 5 g to 60 g Mucuna pruriens or an extract thereof areadministered to a patient. However, the amount particularly depends onfactors such as the body weight, age or disease condition. Therefore, atleast 5 g, at least 10 g, at least 15 g, at least 20 g, at least 25 g,at least 30 g, at least 35 g, at least 40 g, at least 45 g, at least 50g, at least 55 g or at least 60 g of Mucuna pruriens may be appropriatefor achieving the desired therapeutical effect. As the active componentsmay be concentrated in a particular extract, smaller quantities mayalready be sufficient to induce the desired effect in the patient.

The figures show:

FIG. 1: Interpatient means of the plasma levels of L-Dopa followingLD/DC 200/50, 15 g and 30 g Mucuna pruriens. The horizontal axis showstime in hrs:min, the vertical axis the L-Dopa plasma levels in ng/ml.

FIG. 2: The intrapatient means of the plasma levels of L-Dopa followingLD/DC 200/50, 15 g and 30 g Mucuna pruriens. The difference in plasmalevels is presented in percentage difference in L-Dopa plasma levelcompared with the plasma level following LD/DC (=100%).

FIG. 3: The intrapatient means of the plasma levels of 3-O-Methyl-Dopa(3-OMD) following LD/DC 200/50, 15 g and 30 g Mucuna pruriens. Thedifference in plasma levels is presented in percentage difference in3-OMD plasma level compared with the plasma level following LD/DC(=100%).

The invention is illustrated by the following examples:

CLINICAL STUDIES EXAMPLE 1 Patients, Methods and Study Design

Patient Selection:

Patients with idiopathic PD fulfilling the Queen Square Brain Bankcriteria (9) with motor fluctuations and a defined short duration L-Doparesponse (1.5-4 hours) were eligible for inclusion. Clinically relevantpeak dose dyskinesias following each morning dose of their currentmedication were a further pre-requisite. Patients were also required tohave been stable on a fixed dose of treatment for a period of at leastone month prior to starting the study.

Patients were excluded if their current drug regime includedslow-release formulations of L-Dopa, COMT inhibitors, selegiline,anticholinergic drugs, or other drugs that could potentially interferewith gastric absorption (e.g. antacids). Other exclusion criteriaincluded patients with psychotic symptoms or those on antipsychotictreatment patients with clinically relevant cognitive impairment,defined as MMS (Mini Mental State) score of less than 24 (10), risk ofpregnancy, Hoehn & Yahr stage 5 in “off”-status, severe, unstablediabetes mellitus, and medical conditions such as unstablecardiovascular disease or moderate to severe renal or hepaticimpairment. Full blood count, liver and renal function blood tests weretaken at baseline and after completion of the study.

Study Design:

The trial was randomized, double-blind and cross-over in design. Eachpatient was randomized to the order in which either LD/DC or one of thetwo dosages of Mucuna pruriens seed powder preparation were administeredin a single-dose challenge in double-dummy fashion in three consecutivesessions. Randomization was by computer generation of a treatmentnumber, allocated to each patient according to the order of entry intothe study. Study drugs were kept at the pharmacy of the NationalHospital for Neurology and Neurosurgery, London, and dispensed by anindependent pharmacist. The Medicines Control Agency/Department ofHealth, UK, issued an exemption from licenses order for the study drug.The study was approved by the Joint Ethics Committee of UniversityCollege London/University College London Hospitals. All patients gaveinformed consent.

Study Drug:

The Mucuna pruriens seed powder preparation was a light, yellowishpowder, which was manufactured and formulated under Good ManufacturingPractice (GMP) conditions in Germany (Wiewelhove GmbH TM) from raw beanmaterial obtained in India and packed in sachets (unit) of 7.5 grams. Toenhance stability and dissolvability in water, and to improve tasteascorbic acid, tangerine oil, silicium dioxide, saccharine-Na and citricacid, as well as sorbitol and lecithin were added. Matching placebosachets containing powder material with the same consistency, colour andtaste were produced for the study. Quality Assurance Certificates forthe preparation and placebo were obtained from an independent laboratory(LAT GmbH, Munich, Germany). The HPLC-analysis provided demonstrated aL-Dopa content of 4.86% or 250 mg per sachet.

Single Dose Challenges:

Patients were admitted to the hospital for an overnight stay prior tothe challenging session the next morning on three separate occasions atweekly intervals. After withdrawal of all antiparkinsonian medicationfrom midnight the previous day challenges were performed at exactly thesame time in the morning in each patient under fasting conditions withthe exception of black tea or coffee and water.

Patients were randomized to the order of the days on which theyreceived: 200 mg L-Dopa/50 mg carbidopa as capsule formulation plus 4sachets of 7.5 gr placebo powder formulation, or either: 15 g=2 sachetsof Mucuna seed powder (containing 500 mg L-Dopa) plus two sachets ofplacebo powder plus a placebo capsule identical in shape, colour andtaste to the LD/DC capsule, or 30 g=4 sachets of Mucuna seed powder(containing 1000 mg L-Dopa) plus a placebo capsule.

Pharmacokinetic Assessment:

Prior to the challenge a 22 G intravenous catheter was inserted in thepatients forearm. Blood samples of 5 ml each were taken at baseline and15, 30, 45, 60, 75, 90, 105, 120, 140, 160, 180, 210 and 240 minutesafter intake of the medication or until a full “off-state” had beenreached if this occurred earlier than 240 minutes after drug ingestion.Samples were centrifuged immediately at the end of each assessment andstored deep frozen until assayed. Plasma L-Dopa and 3-O-methyl-dopalevels were assessed by high pressure liquid chromatography (HPLC).

On the last assessment additional blood was drawn for routinehematology, blood sugar, liver and renal function.

Clinical Assessment:

Motor function was assessed using UPDRS (United Parkinson's DiseaseRating Scale) motor score and “BrainTest” (11): a tapping test performedwith the patient's more affected hand on the keyboard of a laptopcomputer. These tests were carried out at baseline and then immediatelyfollowing each blood sample until patients had reached their full“on”-stage. Thereafter at 3 intervals of 20 minutes, and 30 minutesintervals until 240 minutes after drug administration, or until patientshad reached their baseline “off”-status, whichever came first. Oncepatients had reached their full “on”-state, video recordings wereperformed three times at 20 minutes intervals. As certain mental andmotor tasks have been shown to increase dyskinesia (12), the followingtasks were chosen to be carded out during each video session:

1. Sitting still for 1 minute

2. Performing mental calculations

3. Putting on and buttoning a coat.

4. Picking up and drinking from a cup of water

5. Walking

Videotapes were rated independently by two blinded raters using modifiedversions of the Goetz Rating Scale and the Abnormal InvoluntaryMovements Scale (AIMS) to document a possible increase in drug induceddyskinesia.

Modified versions were applied in that global rating and facial muscleswere excluded for AIMS, while for Goetz phenomenological rating wasexcluded and only choreatic movements were counted.

The actual occurrence and severity of dyskinesia was measured with aDyskinesia Monitor, a small ambulatory electronic device developed toobjectively assess dyskinesias over prolonged periods of time (13). Thedevice is taped to the patients shoulder on their more affected side.The monitor records during the entire time of the challenging sessionand provides an absolute value being the result of the frequency andseverity of occurring dyskinesias.

Blinding:

Randomization information was kept in a blinded format with the companythat had manufactured and supplied the active drugs and the matchingplacebos. Emergency envelopes with the randomization code were also keptwith the head pharmacist at the National Hospital for Neurology andNeurosurgery, London. Blinding was maintained until after the databasewas locked.

Statistics and Data Analysis:

The double-blind trial was powered to detect a difference of 25% betweenAIMS scores on treatment, which was considered a clinically relevantchange based on previous publications (13) and on clinical judgment.Power calculation showed that eight patients completing the treatmentarms were required to obtain a power of 80% at the 5% significancelevel. All data sets were assessed and means/medians were compared usingWilcoxon's non-parametric signed rank-test or paired-samples t-test, asappropriate. Both inter- and intra-patient data have been analyzed. Thetwo video raters' AIM and Goetz scores were combined for analysis.Interrater reliability was assessed using kappa analysis, weightedaccording to how close agreement between the raters was (StataStatistical Software Release 6.0).

Patient Population:

Nine patients (five women and four men) were enrolled in the study. Onepatient dropped out due to transient vomiting during ingestion of thefirst study medication (30 g Mucuna). Since absorption was not likely tohave taken place in this patient, this patient was excluded from furtherevaluation. Eight patients completed the three sessions of this study.Patients mean age was 62.2 years (range 50-72). Mean disease durationwas 12.4 years (range 7-17). Mean Hoehn & Yahr stage (in “off”-stage)was 3.5 (range 2.5-4). Patients took a mean daily L-Dopa dose of 572 mgprior to the trial. Other antiparkinsonian medication taken by anypatient were amantadine in two (200 mg), pergolide in three (mean, 3.2mg), and ropinirole (18 mg), cabergoline (6 mg) and pramipexole (1.4 mg)in one patient each.

EXAMPLE 2 Pharmacokinetic Results

In FIG. 1 the mean serum L-Dopa levels are presented. The mean C_(max)for both 15 g and 30 g were significantly higher than for LC/DC (+57%and +163%, respectively). T_(max) was reached for 15 g Mucuna after 30minutes, for 30 g Mucuna after 45 minutes and for LD/DC after 90minutes. There were no significant differences in T_(1/2) among thethree study drugs. The mean L-Dopa plasma levels of 15 g Mucuna startedto decline below the level of LD/DC after approximately 80 minutes. Inview of interpatient variability and corresponding standard deviations(at C_(max) LD; 1600 ng/ml, 15 g: 5962 ng/ml, 30 g: 7213 ng/ml) theintrapatient means are presented in FIG. 2 the differences between hestudy drugs expressed as percentage difference from LD/DC (LD/DC=100%).The peak difference for both 15 g and 30 g Mucuna is reached after 15minutes (+1659% and +3155%, respectively).

Also on the basis of intrapatient means the L-Dopa plasma level of 15 gMucuna declines under the level of LD/DC after 90 minutes, whilst 30 gMucuna maintains a difference of +88% to +113%. The means of cumulativeintrapatient plasma relations (intrapatient AUC differences, AUCLD/DC=100%) demonstrate a difference of +31% for 15 g and +159% for 30 gMucuna. Although values tended to be higher in patients administeredLD/DC, 3-OMD AUC values were not significantly different between thethree study regimens. The intrapatient means for 3-OMD (FIG. 3) did alsonot show a difference in the plasma levels for LD/DC and 30 g Mucuna,but for 15 g Mucuna a steady decline below the levels of LD/DC wasnotable, which reached a difference of −36% after 240 minutes. Thisdifference was significant (p=0.009 at 240 minutes).

EXAMPLE 3 Results of the Clinical Assessment

Results and statistical significance of differences are shown in table 1and 2. Time to full “on” status was 23 minutes (33.4%) shorter on 15 gMucuna than on LD/DC, and 34 minutes (49.5%) shorter on 30 g Mucuna.Time to beginning of switching on was reduced by 27 minutes (49%) on 15g and by 32 minutes (58%) on 30 g Mucuna. These differences were highlysignificant.

TABLE 1 Clinical assessments; measures of parkinsonism on LD/DC, 15 gand 30 g Mucuna pruriens. LD/DC 15 g Mucuna 30 g Mucuna Diff. LD/DCDiff. LD/DC (SD) (SD) (SD) vs 15 g Muc vs 30 g Muc Mean interpatientvalues: UPDRS baseline 49.8 (12.7) 49.5 (15.3) 46.9 (10.7) n.s. p =0.046 BRAIN baseline 47.4 (11.0) 44.0 (12.6) 45.0 (13.6) n.s. n.s. BestUPDRS “on” 15.4 (8.0) 15.5 (7.6) 15.5 (8.5) n.s. n.s. Best BRAIN “on”75.9 (18.4) 78.5 (21.3) 79.1 (15.0) n.s. n.s. Mean intrapatient values:Best UPDRS “on” 0 0.125 0.125 n.s. n.s. Best BRAIN “on” 0 +2.5 +3.125n.s. n.s. Mean interpatient values*: Time to full “on” 68.5 (29.0) 45.6(30.4) 34.6 (13.6) p = 0.035 p = 0.021 Time to beginning of “on” 54.6(24.5) 27.8 (14.1) 23.0 (11.5) p = 0.012 p = 0.012 Duration of full “on”167.4 (55.3) 147.3 (30.5) 204.5 (55.1) n.s. p = 0.021 Mean intrapatientnormalized values: Duration of full “on” 100% 99% 128% *values inminutes. SD = Standard deviation. LD/DC = L-Dopa/carbidopa. BRAIN seeMethods. UPDRS refers to motor cores.

TABLE 2 Clinical assessments; dyskinesia measures on LD/DC, 15 g and 30g Mucuna pruriens. LD/DC 15 g Mucuna 30 g Mucuna Diff. LD/DC Diff. LD/DC(SD) (SD) (SD) vs 15 g Muc vs 30 g Muc Mean AIMS score 8.0 (5.0) 8.6(4.9) 8.0 (5.1) p = 0.36 p = 0.75 Mean Goetz score 2.0 (0.6) 2.1 (0.6)1.9 (0.5) p = 0.07 p = 0.34 Dyskinesia Monitor Index: Mean interpatientindex 41.7 (12.1) 39.7 (11.1) 37.4 (6.1) na na Mean intrapatient index 0−1.98 −4.32 p = 0.16 p = 0.27 SD = Standard Deviation LD/DC =L-Dopa/carbidopa.

On an interpatient mean basis the duration of full “on”-status was 37minutes (22%) longer on 30 g Mucuna compared with LD/DC. This differencewas significant (p=0.021). The duration on 15 g mucuna was somewhatshorter than with LD/DC; by 20 minutes (12%). This difference was notsignificant supported by the normalized intrapatient evaluation (mean of% difference in individual patients) in which 15 g Mucuna reached 99% ofthe “on” time duration time of LD/DC. Although a difference in UPDRSscores at baseline reached significance for 30 g Mucuna versus LD/DC inthe mean interpatient analysis, best UPDRS motor scores and tappingspeed (BRAIN) when “on” did not differ significantly among the threestudy drugs.

The interrater reliability with respect to the rating of videorecordings was satisfactory to good: Weighted kappa was 0.45 (p<0.0001)for Goetz scores (Spearman's rank 0.87, p<0.0001) and 0.62 (p<0.0001)for AIMS scores (Spearman's rank 0.97, p<0.0001). No significantdifferences in ratings were found among the study drugs. Since of onepatient values were not listed due to a dislocation of the device duringone session, the results with the Dyskinesia Monitor can only beevaluated for 7 patients. In spite of the significant differences in LDplasma levels, both the inter- and intrapatient DMI values did notdiffer significantly, but a dose dependent reduction in index values forMucuna compared with LD/DC was recorded. Whether this reduction has aclinical basis needs to be further evaluated in view of the results withAIMS and Goetz.

EXAMPLE 4 Safety and Tolerability

Apart from the patient who dropped out due to vomiting during the intakeof 30 g Mucuna and placebos, other adverse events were: mild andshort-lasting nausea occurring in two patients on LD/DC and: in twopatients on 30 g Mucuna, mild gastric pain in one patient on LD/DC, andmild dizziness in one patient each on LD/DC and on 15 g Mucuna. Totalepisodes were therefore 4 for LD/DC, 1 for 15 g and 2 for 30 g Mucuna.No clinically relevant changes in hematology and biochemistry parameterswere observed.

IN VITRO STUDIES EXAMPLE 5 Material and Methods of In Vitro Studies

Experimental Design:

Thirteen (13) different extracts of Mucana Pruriens were tested on thesurvival of primary cell cultures of mesencephalic neurons. Cellcultures were prepared from embryonic rodent mesencephalon on the14^(th) day of gestation. Three or four concentrations of each extractwere tested in triplicate in each paradigm, according to a previouslypublished method (Mytilineou et al 1997, 1998). All experiments havebeen performed in a blinded fashion.

Extracts of Mucana Pruriens were tested for their effect on thefollowing conditions:

-   -   (I) Survival of cultured dopaminergic neurons    -   (II) Survival of cultured dopamine neurons following exposure        to:        -   (a) depletion of GSH by buthionine sulfoximine (3            concentrations)        -   (b) exposure to MPP⁺ (3 concentrations)            Analysis of Data:

The effect of extracts of Mucana Pruriens on the survival of dopamineneurons were assessed by measuring:

-   -   (a) the uptake of dopamine as a measure of the number of        dopamine terminals and an index of the number of surviving        dopamine neurons.    -   (b) MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium        bromid) reduction as a measure of cell survival    -   (c) LDH (lactate dehydrogenase) release as a measure of number        of survival cells

These techniques are well known to the person skilled in the art andhave been published previously (Mytilineou et al 1997, 1998).

Miscellaneous Material: Pregnant rats are purchased from Taconic Farms(Germantown, N.Y.). Minimum essential medium (MEM) is obtained fromGIBCO (Grand Isand, N.Y.), horse serum from Gemini (Calabasas, Calif.),and NU serum from Becton Dickinson (Bedford, Mass.). The Vectastain ABCKit is form Vector Laboratories (Burlingame, Calif.). Other chemicalsare purchased form Sigma (St. Louis, Mo.).

Cell Culture:

Mesencephalic cultures are prepared from rat embryos on gestational day14 as previously described (Mytilineou et al 1993). Dissociated cellsare plated on poly-l-omithine (0.1 mg/ml)-coated dishes (35 mm indiameter, Falcon) at a density of 10⁵ cells/cm². The feeding mediumconsists of MEM with 30 mM glucose, 2 mM glutamine, 10% horse serum and10% NU serum (which contains 25% fetal calf serum and other additions).

Assay for Dopamine Uptake:

[³H]DA uptake is determined as previously described (Hou et al. 1996).In brief, cultures are washed twice to remove residual drugs andincubated for 15 min in [³H]DA (0.5 Ci/ml, 21.4 Ci/mmol). After tworinses and a 5 min incubation with fresh buffer, the accumulated [³H]DAis extracted in 1 ml of 95% ethanol, added to 10 ml of Exoscint A andcounted in a scintillation spectrometer.

MTT Assay:

Cell viability was determined by the MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromid) reductionassay,as described previously (Han et al., 1996). In brief, 50 μl of a 5mg/ml solution of MTT was added to each cell culture well containing 0.5ml medians. After 3 hours incubation at 37° C. the medium was removedcarefully and the formazan crystals formed were dissolved in 1 mlisopropyl alcohol by gently shaking of the plate. Absorbance wasdetermined at 570 nm in a microplate reader.

LDH Assay:

A modification of the method by Bergmeyer et al. (1963) was used todetermine LDH activity in the culture medium and the cells. Culturemedium was collected, centrifuged to remove debris and frozen at −80° C.until assay. Cells were collected in 1.0 ml of 50 mM potassium phosphatebuffer at pH 7.2, sonicated in the cold for 10 s and frozen at −80° C.100 μl of supernatant and 100μl of NADH (1.2 mg/ml H₂O stock) were addedto 800 μl of buffer and the samples were vortex-mixed. 250 μl aliquots(triplicates) were placed into 96-well plates at room temperature andthe reaction was initiated by addition of 25 μl of sodium pyruvate (0.35μg/ml H₂O stock). The rate of disappearance of NADH was measured at 340nm on a plate reader (Spectramax™, Molecular Devices Corporation,Sunnyvale Calif.).

Statistical assessment:

For multiple comparisons, statistical analysis were carried out using anANOVA followed by Tukey's or Dunnett's test. Significance between groupswas tested with an independent two-tailed t-test.

Composition and Properties of Extracts:

-   1. M-HX1299 (50 mg): 200 g pulverised seed material of Mucana    pruriens were shaken at room temperature in 200 ml n-hexane for 18    hours. After filtration, the material was further washed with 100 ml    n-hexane and filtered. The filtrates were collected and the solvent    distilled off to obtain a yellowish oil.-   2. M-AC1299 (50 mg): The residue of the n-hexane extraction (was    above), was shaken for 18 hours at room temperature in acetone    (200 ml) and filtered. The residue was extracted once more with 200    ml acetone by further shaking for 18 hours and filtered. The residue    was washed with acetone (100 ml) and filtered. After pooling, the    filtrates were evaporated by distillation under reduced pressure    yielding a yellowish mass.-   3. M-W-EL1299 (50 mg): The residue (obtained from the above    extractions, ca. 96 g) was shaken for 18 hours at room temperature    in 500 ml of a mixture of water-EtOH, 1:1 with 0.5% ascorbic acid.    The solvent was filtered and concentrated under reduced pressure at    a temperature of 35° C. Above extraction procedure was repeated four    times. After concentration, the filtrates were collected and the    solvent removed under vacuum to get the solid mass.-   4. M-CH1299 (50 mg): 100 g of the pulverised seed s of M. pruriens    were shaken for 18 hours at room temperature in EtOH (100 ml). After    filtration, the residue was again shaken for 18 hours and filtered.    The process was repeated for a total of four extractions. The    filtrates, were concentrated and pooled together to get the extract.-   5. M-EL0100 (50 mg): 20 g of the pulverised seeds of M. pruriens    were shaken for 18 hours at room temperature in EtOH (100 ml). After    filtration, the residue was again shaken for 18 hours and filtered.    The process was repeated for a total fo four extractions. The    filtrates were concentrated and pooled together to get the extract.-   6. M-W0100 (50 mg): The residue obtained from the ethanol    extraction, as stated above, was further shaken for 18 hours at room    temperature in demineralised water and filtered. The extraction was    repeated for three times more. The filtrates were pooled together    and water distilled off, after passing SO₂ to prevent the oxidation    of L-DOPA. The solid sticky extract was thus obtained.-   7. M-ML0100 (50 mg): 10 g of the pulverised seeds of M. pruriens    were shaken for 18 hours at room temperature in methanol (50 ml) and    filtered. The extraction of the residue was followed two more times    with methanol and filtered. The filtrates were pooled together and    the solvent distilled off to yield the semi-solid mass.-   8. M-BL0100 (50 mg): 10 g of the pulverised seeds of M. pruriens    were shaken for 18 hours at room temperature in n-butanol (50 ml)    and filtered. The extraction of the residue was followed two more    times with n-butanol and filtered. The filtrates were pooled    together and the solvent distilled off. The oily extract was thus    obtained.-   9. M-PL0100 (50 mg): 10 g of the pulverised seeds of M. pruriens    were shaken for 18 hours at room temperature in n-propanol (50 ml)    and filtered. The extraction of the residue was again followed two    more times with n-propanol and filtered. The filtrates were pooled    together and the solvent distilled off to get an oily mass.-   10. M-ACPL0800: The M. pruriens seed powder was defatted twice or 18    hrs at room temperature with acetone by shaking the powder each with    200 ml acetone, the combined solvent evaporated at reduced    temperature and the residue extracted for 18 hrs at room temperature    with 500 ml n-propanol and then evaporated to dryness. This extract    contains traces (negligible) amounts of L-DOPA (in TLC detectable    after enrichment only).-   11. MWEL0700: The M. pruriens seed powder was not defatted with    acetone, but directly extracted with a 1:1 mixture of water:ethanol    without ascorbic acid addition by shaking 100 g powder with 50.0 ml    of this solvent mixture for 18 hrs at room temperature. The solvent    was evaporated at reduced temperatur to dryness. The extract    contains L-DOPA in traces only.

SOLUBILITY OF THE EXTRACTS  1. M-HX1299: 6.5 mg in 0.5 ml DMSO + 0.5 mldist. water  2. M-AC 1299: 2.0 mg in 6 drops of DMSO + 0.2 ml dist.water  3. M-W-EL 1299: 5.0 mg in 0.1 ml dist. Water  4. M-CH1299: 10.0mg in 0.3 ml DMSO + 0.8 ml dist. water  5. M-EL0100: 3.0 mg in 2 dropsDMSO + 1.0 ml dist. water  6. M-W0100: 7.0 mg in 0.4 ml warm (60° C.)dist. water  7. M-ML0100: 4.0 mg in 0.3 ml dist. water  8. M-BL0100: 7.0mg in 4 drops DMSO + 0.4 ml dist. water  9. M-PL0100: 5.0 mg in 4 dropsDMSO + 0.4 ml dist. water 10. LAT543 soluble in water in variousconcentrations, light opalescent 11. LAT543-0 soluble in water invarious concentrations, light opalescent

L-DOPA COMPOSITION OF THE EXTRACTS  1. M-HX1299: No L-DOPA content.  2.M-AC1299: No L-DOPA content.  3. M-W-EL1299: High L-DOPA content.  4.M-CH1299: No L-DOPA content.  5. M-EL0100: Moderate L-DOPA content.  6.M-W0100: High L-DOPA content.  7. M-ML0100: Moderately high L-DOPAcontent  8. M-BL0100: Moderate L-DOPA content.  9. M-PL0100: Trace ofL-DOPA content. 10. M-ACPL0800 Trace of L-DOPA content. 11. MWEL0700Trace of L-DOPA content. 12. LAT543 No L-DOPA content 13. LAT543-0 NoL-DOPA content

EXAMPLE 6 Extraction Procedure of Mucuna Pruriens Seeds, FractionatedExtraction

Mucuna Pruriens extracts were generated by performing steps 1 to 7 ofthe following protocol:

Extraction Procedures of Mucuna pruriens Seeds (Batch No. MU 99001)

-   1. Hexane extract (no. M-HX1299): 200 g pulverised seed material of    Mucuna pruriens were shaken at room temperature in 200 ml n-hexane    for 18 hrs. After filtration, the material was further washed with    100 ml n-hexane and filtered. The filtrates were collected and    solvent distilled off to obtain a yellowish oily liquid (5.5 g) in a    yield of 2.75% (w/w). The 50 mg extract gave clear solution in 1 ml    DMSO (5% v/v).-   2. Acetone extract (no. M-AC1299): The residue of the n-hexane    extraction, was shaken for 18 hrs. at room temperature in acetone    (200 ml) and filtered. The residue was extracted once more with 200    ml acetone by further shaking for 18 hrs. The residue was washed    with acetone (100 ml) and filtered. After pooling, the filtrates    were evaporated by distillation under reduced pressure yielding an    yellowish mass (2.02 g) to a yield of 1.0% (w/w).-   3. Water-Ethanol (1:1) extract (M-W-EL1299): The residue (ca. 96 g)    was shaken for 18 hrs. at room temperature in 500 ml of a mixture of    water, EtOH, 1:1 with 0.5% ascorbic acid. The solvent was filtered    and concentrated under reduced pressure at a temperature of 35° C.    Above extraction procedure was repeated four times. After    concentration, the filtrates were collected and the solvent reduced    to one tenth and kept at 2-4° C. for 24 hour. The crystallized    matter was filtered and filtrate was kept for another 24 hours at    2-4° C. The crystallized matter was again filtered and taken    together to get 1.75 g crystals in a yield of 1.75%. The filtrate    was evaporated to dryness to get a solid mass (22.51 g) with 1.78 g    as crude L-DOPA obtained after crystallization.-   4. Chloroform extract (M-CH1299): 100 g of the pulverised seeds    of M. pruriens were shaken for 18 hrs. at room temperature in 1.7%    ammoniated chloroform (300 ml). The extract was filtered and the    extraction was repeated three times. The concentrated extract was    washed with water (100 ml) and further concentrated to afford 4.0 g    extract in a yield of 4.0 (w/w).-   5. Ethanol extract (M-EL0100): 20 g of the pulverised seeds of M.    pruriens were shaken for 18 hrs. at room temperature in EtOH (100    ml). After filtration, the residue was again shaken for 18 hrs. and    filtered. The process was repeated for a total of four extractions.    The filtrates were concentrated and pooled together to get 1.34 g    extract. The extract was tested for the presence of L-DOPA by TLC,    positively.-   6. Aqueous extract (M-W0100): The residue abtained from the ethanol    extraction, as stated above, was further shaken for 18 hrs. at room    temperature in demineralised water and filtered. The extraction was    repeated for three times more. The filtrates were pooled together    and water distilled off, after passing SO₂ to prevent the oxidation    of L-DOPA. The extract (4.68 g) was tested for the presence of    L-DOPA.-   7. Acetone extract (M-AC0100): 10 g of the pulverised seeds of M.    pruriens were shaken for 18 hrs. at room temperature in acetone (50    ml). After filtration, the residue was again extracted with acetone.    The extraction was repeated three times more. The filtrates were    pooled together and the solvent distilled off. The extract (0.37 g)    was used for the presence of phosphatides.

EXAMPLE 7 Effect of Extracts on Primary Dopaminergic Cultures

Mesencephalic cultures were treated with different concentrations ofextracts for 7 days. [³H]Dopamine uptake was measured as an index ofdopamine neuron survival and growth. This is a quantitative measurementthat reflects the number of dopamine neurons and terminals.

The results of the extracts on native dopaminergic cultures are shown intable 3. Three extracts stimulated at a dose of 0.05 μg/ml. However,only one extract, M-PL0100, stimulated at all doses and significantlyincreased dopamine uptake in the cultures after one week of treatment.The effect was close to a 2-fold increase. However, the SEM(SEM=Structural Equation Modeling, a comprehensive statistical approachto testing hypotheses about relations among observed and latentvariables (measured variables and unmeasured constracts) was quite high.Notably, no dose response was observed, possibly due to maximumeffectiveness of the lowest concentration used (0.05 μg/ml). M-EL0100and M-BL0100 showed a significant increase in uptake only at 0.05. μg/mland LAT543-0 only at 50 μg/ml.

TABLE 3 Effect of Mucana pruriens extracts on the growth of dopamineneurons Compound [³H]Dopamine Uptake (% of Control) (μg/ml) 0.05 0.5 550 M-HX1299 124.9 ± 10.2  106.0 ± 11.2  92.9 ± 9.7 100.5 ± 13.3 M-AC1299115.4 ± 9.0   95.4 ± 8.9  93.1 ± 9.3 121.7 ± 7.1  M-W-EL1299 127.8 ±10.4 111.1 ± 6.8 109.1 ± 7.5 100.5 ± 12.8 M-CH1299 129.5 ± 18.1  115.8 ±12.7 105.3 ± 8.1 101.9 ± 9.0  M-EL0100  184.9 ± 20.3*  146.5 ± 14.9 127.8 ± 12.5 140.8 ± 12.5 M-W0100 120.8 ± 9.4  102.7 ± 8.9  103.3 ±13.3  86.3 ± 11.2 M-ML0100 122.9 ± 13.4 119.5 ± 8.3  115.3 ± 13.1 106.4± 10.0 M-BL0100  139.1 ± 19.4* 108.3 ± 9.9  89.4 ± 6.7 130.1 ± 9.3 M-PL0100  206.3 ± 30.4*  187.7 ± 20.3*  170.1 ± 21.3*  191.6 ± 24.5*LAT543 135.1 ± 26.5  108.6 ± 14.2  117.6 ± 16.3 105.6 ± 10.2 LAT543-0139.1 ± 19.4 117.9 ± 8.6  120.4 ± 12.4  156.2 ± 14.6* MACPL0800 — — — —MWEL0700 — — — — *p < 0.05 ANOVA followed by Dunnett multiple comparisontest

EXAMPLE 8 Effect of Extracts in Protecting Mesencephlic Cultures fromthe Toxic Effects of MPP³⁰

Mesencephlic cultures were treated with 5 μM MPP+ for 24 hours in theabsence or presence of different concentrations of extracts.[³H]Dopamine uptake was measured 48 hours after removing MPP⁺(table 4).Four compounds protected dopamine neurons from MPP+ toxicity(M-W-EL1299; M-W0100 and MWEL0700). All were effective at aconcentration of 50 μg/ml.

TABLE 4 Effect of Mucuna pruriens extracts on the toxicity of MPP+ todopamine neurons Extract [³H]Dopamine Uptake (% of no MPP+) (μg/ml) 00.5 5 50 M-HX1299 25.7 ± 2.5 27.4 ± 2.6 30.1 ± 3.7 27.3 ± 2.5 M-AC129931.4 ± 3.6 37.9 ± 4.4 35.5 ± 3.2 34.9 ± 2.7 M-W-EL-1299 25.5 ± 2.5 31.3± 3.8 33.2 ± 3.3 63.1 ± 5.7 M-CH1299 31.3 ± 1.7 32.6 ± 3.1 29.1 ± 3.134.3 ± 2.2 M-EL0100 34.6 ± 6.0 33.0 ± 5.3 40.0 ± 5.8 47.2 ± 8.0 M-W010039.1 ± 3.7 52.0 ± 4.1 50.8 ± 2.5 59.6 ± 4.6 M-ML0100 28.8 ± 2.0 23.7 ±1.2 32.9 ± 2.4 50.7 ± 2.4 M-BL0100 36.0 ± 4.0 32.8 ± 4.6 34.7 ± 4.9 36.5± 3.4 M-PL0100 30.6 ± 4.6 31.6 ± 3.9 31.2 ± 4.7 40.4 ± 4.6 LAT543 30.2 ±3.0 31.8 ± 3.9 34.7 ± 7.2 30.4 ± 4.6 LAT543-0 28.9 ± 1.4 28.3 ± 2.6 27.3± 2.1 33.7 ± 1.1 MACPL0800 — — — — MWEL0700 37.4 ± 2.8 36.4 ± 3.2 49.1 ±3.4 78.6 ± 8.6 *Significance p < 0.05 or greater; ANOVA followed byTukey-Kramer multiple comparisons test.

EXAMPLE 9 Effect of Extracts in Protecting Mesencephalic Cultures fromthe Toxic Effects of BSO

Mesencephalic cultures were treated with 10 or 50 μM BSO for 72 hours toreduce GSH levels and cause oxidative damage. The extracts were added atthe same time as the BSO. The MTT assay was performed to determine cellviability (table 5). As GSH depletion is toxic to all cells, theprotection by the extracts in this assay is not necessarily restrictedto dopamine neurons.

The LDH assay (table 6) was performed in the medium collected from thecultures and is a measure of cell viability. As this is a non-specificassay, protection by the extracts may not be restricted to dopamineneurons. In the studies, protection against MPP+ toxicity with both theMTT and LDH methods was observed for M-W-EL1299; M-ML0100, and MWEL0700.

TABLE 5 Effect of Mucuna pruriens extracts on toxicity of GSH depletion(MTT assay) MTT Reduction (absorbance at 550) BSO (μM) 0 10 50 Extract(50 μg/ml) − + − + − + M-HX1299 1.60 ± 0.01 1.59 ± 0.03 1.31 ± 0.04 0.23± 0.05*** 0.76 ± 0.04 0.05 ± 0.01*** M-AC1299 1.55 ± 0.02 1.58 ± 0.021.42 ± 0.06 0.15 ± 0.02*** 0.83 ± 0.05 0.03 ± 0.00*** M-W-EL1299 1.68 ±0.11 1.74 ± 0.08 1.52 ± 0.07 1.77 ± 0.09   1.02 ± 0.06 1.81 ± 0.11  M-CH1299 1.88 ± 0.03 1.95 ± 0.02 1.10 ± 0.12 0.04 ± 0.00*** 0.55 ± 0.070.01 ± 0.00*** M-EL0100 1.97 ± 0.03 1.95 ± 0.03 1.36 ± 0.02 0.81 ±0.04*  0.65 ± 0.09 0.11 ± 0.02*** M-W0100 1.26 ± 0.01 1.14 ± 0.04 1.05 ±0.06 0.70 ± 0.02*  0.66 ± 0.07 0.54 ± 0.05   M-ML0100 1.81 ± 0.05 1.84 ±0.06 1.48 ± 0.07 1.80 ± 0.05**  0.84 ± 0.08 1.83 ± 0.05*** M-BL0100 1.22± 0.03 1.32 ± 0.00 1.17 ± 0.06 0.83 ± 0.12   0.91 ± 0.15 0.12 ± 0.02***M-PL0100 1.12 ± 0.02 1.07 ± 0.02 1.04 ± 0.02 0.31 ± 0.05*** 0.64 ± 0.040.03 ± 0.01*** LAT543 1.34 ± 0.05 1.55 ± 0.01 1.20 ± 0.17 0.82 ± 0.16** 0.67 ± 0.16 0.08 ± 0.01*  LAT543-0 1.67 ± 0.02 1.68 ± 0.02 1.24 ± 0.070.49 ± 0.03*** 0.52 ± 0.05 0.09 ± 0.01*** MACPL0800 1.72 ± 0.03 1.64 ±0.03 1.20 ± 0.10 0.30 ± 0.04*** 0.56 ± 0.10 0.05 ± 0.02*** MWEL0700 1.65± 0.03 1.73 ± 0.02 1.04 ± 0.05 1.71 ± 0.02*** 0.61 ± 0.03 1.72 ± 0.04***

TABLE 6 Effect of Mucuna pruriens extracts on toxicity of GSH depletion(LDH assay). The stars in the tables relate to the legends. (p-values).LDH released BSO (μM) 0 10 50 Extract (50 μg/ml) − + − + − + M-HX12997.8 ± 0.2 8.0 ± 0.4 20.8 ± 0.8 77.9 ± 3.3*** 48.0 ± 1.7 75.0 ± 4.6***M-AC1299 6.9 ± 0.3 6.6 ± 0.2  9.1 ± 0.8 53.7 ± 4.7*** 37.1 ± 1.9 60.6 ±0.6*** M-W-EL1299 5.2 ± 0.4 4.9 ± 0.9 11.9 ± 1.7  4.8 ± 0.9*** 27.9 ±5.0  4.8 ± 1.0*** M-CH1299 6.9 ± 0.2 6.4 ± 0.2 37.6 ± 4.7 66.2 ± 1.2***52.1 ± 2.2 69.5 ± 2.0*  M-EL0100 7.2 ± 0.2 6.4 ± 0.1 28.7 ± 1.2 49.4 ±0.6*** 50.2 ± 1.4 67.7 ± 1.7*  M-W0100 3.4 ± 0.1 5.9 ± 0.6 15.0 ± 2.219.4 ± 0.9   27.6 ± 2.4 27.5 ± 2.0   M-ML0100 8.4 ± 0.4 8.0 ± 0.3 20.9 ±1.9  7.7 ± 0.4*** 47.7 ± 2.8  8.7 ± 0.3*** M-BL0100 4.2 ± 0.2 4.6 ± 0.4 8.7 ± 1.1 23.6 ± 4.2**  20.4 ± 4.5 43.9 ± 1.4*** M-PL0100 7.3 ± 0.1 7.4± 0.5 10.0 ± 0.5 47.3 ± 2.8*** 21.4 ± 5.4 54.8 ± 1.1*** LAT543 3.9 ± 0.24.5 ± 0.3 13.6 ± 5.8 29.4 ± 3.9**  37.2 ± 5.6 45.1 ± 1.0   LAT543-0 7.2± 0.8 7.5 ± 0.7 28.1 ± 3.9 64.9 ± 2.1*** 59.9 ± 2.9 76.3 ± 2.7* MACPL0800 7.0 ± 0.6 6.0 ± 0.5 31.4 ± 6.2 64.7 ± 1.8*** 57.0 ± 5.0 70.8 ±1.0*  MWEL0700 7.3 ± 0.5 7.3 ± 0.6 41.6 ± 2.1  7.5 ± 0.8*** 57.2 ± 1.6 8.9 ± 0.7***

Lactate dehydrogenase (LDH) is a cellular enzyme which is released fromdamaged or dying cells. Therefore, LDH values increase in case ofcellular distress/damage. A strong increase after BSO addition is to beexpected to the resulting GSH depletion. Without BSO the extracts didnot increase LDH, indicating no toxic effects of the extracts themselfs.With BSO and with increasing dose LDH levels increase strongly with theexception of 3 extracts where LDH is maintained at the o levelindicating strong protection. Remarkably, a number of extracts show arise of LDH levels, indicating increased toxicity. All those extractscontain DMSO which in high concentrations may be toxic. It is likelythat in this model DMSO potentiated the BSO toxicity. A similarphenomenon can be seen in table 5.

Something similar may be the case in the stimulation model (table 3).Three extracts stimulate with a dose of 0.05 μg/ml. Only M-PLO100 withall doses. All three extracts are alcohol extracts, however, only PL0100does not contain DMSO and only a trace L-Dopa, the other two extractscontain DMSO and moderate L-Dopa concentration. Thus, it is possiblethat with increasing doses DSMO and/or L-Dopa (alone or in synergy) havea toxic effect and thereby undo the stimulatory effect.

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1. A method of preparing an extract or extract fraction of Mucunapruriens seeds comprising: extracting a seed of Mucuna pruriens withn-hexane to provide a first extract solution; filtering the firstextract solution to provide a first filter retentate; extracting thefirst filter retentate with acetone to provide a second extractsolution; filtering the second extract solution to provide a secondfilter retentate; extracting the second filter retentate with anapproximately 1:1 mixture of water and ethanol containing approximately0.5% ascorbic acid to provide third extract solution; filtering thethird extract solution to provide a third filter retentate; repeatingthe extraction with an approximately 1:1 mixture of water and ethanolcontaining approximately 0.5% ascorbic acid at least four times usingthe third through a sixth filter retentate to provide a fourth throughseventh extract solutions; pooling at least two of the extract solutionsto provide a pooled extract solution; and concentrating the pooledextract solution to form an extract or extract fraction containing atleast one pharmaceutically active component, substance, fraction, ormixture thereof.
 2. The method of claim 1, further comprisingsolubilizing the extract or extract fraction in a solvent comprisingDMSO, distilled water, or a mixture thereof.
 3. The method of claim 1,further comprising formulating the extract or extract fraction in a formselected from the group consisting of comminuted form, as granules,powder, precipitate, dried extract, exudate, and any combinationsthereof.
 4. The method of claim 1, further comprising formulating theextract or extract fraction for oral application, topical application orparenteral application.
 5. The method of claim 1, further comprisingpreparing a formulation selected from the group consisting of aninfusion solution, an injection solution, an orally administrable form,a gelatin-capsule, a tablet, a controlled release tablet, a granulate, afood supplement, an enema, and any combinations thereof.
 6. The methodof claim 1, wherein the component, substance, fraction, or mixturethereof does not contain a pharmaceutically effective amount of L-dopa.7. The method of claim 1, wherein the extract or extract fractionsfurther comprises at least one additional pharmaceutically active agent.